WO2018191771A1 - Detection and treatment of congenital malformations - Google Patents

Detection and treatment of congenital malformations Download PDF

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WO2018191771A1
WO2018191771A1 PCT/AU2018/000055 AU2018000055W WO2018191771A1 WO 2018191771 A1 WO2018191771 A1 WO 2018191771A1 AU 2018000055 W AU2018000055 W AU 2018000055W WO 2018191771 A1 WO2018191771 A1 WO 2018191771A1
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nad
nicotinamide
acid
synthesis
adp
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PCT/AU2018/000055
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French (fr)
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Sally Lewers DUNWOODIE
Duncan Burnaby SPARROW
Roland Otto STOCKER
Hongjun SHI
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Victor Chang Cardiac Research Institute
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Priority claimed from AU2017901409A external-priority patent/AU2017901409A0/en
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Publication of WO2018191771A1 publication Critical patent/WO2018191771A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the technology relates to methods of detecting defects in nicotinamide adenine dinucleotide (NAD) synthesis that cause congenital malformations.
  • the technology also relates to methods of treating congenital malformations caused by defects in NAD synthesis.
  • V vertebral defects
  • A anal atresia
  • C cardiac malformations
  • TE tracheoesophageal fistula with esophageal atresia
  • R renal dysplasia
  • L limb anomalies
  • the kynurenine pathway or de novo NAD synthesis pathway is the metabolic pathway which is responsible for the production of nicotinamide adenine dinucleotide (NAD) from the essential amino acid tryptophan.
  • NAD nicotinamide adenine dinucleotide
  • kynurenine pathway are 3-hydroxyanthranilate 3,4-dioxygenase (HAAO) and
  • kynureninase KYNU
  • HAAO catalyzes the synthesis of quinolinic acid from 3- hydroxyanth rani lie acid.
  • KYNU catalyzes the hydrolysis of 3-hydroxy-L-kynurenine to 3- hydroxyanth rani lie acid and L-alanine.
  • Another pathway by which NAD is produced is the NAD salvage pathway. In this pathway NAD is produced from nicotinamide and nicotinamide riboside. NAD can also be produced by the Preiss-Handler pathway. In this pathway NAD is produced from nicotinic acid. Some of these compounds are found in the diet but all are produced within cells and by metabolism of cellular NAD.
  • the NAD salvage pathway is essential in humans because there is a high cellular demand for NAD.
  • the rate-limiting enzyme in the NAD salvage pathway is nicotinamide phosphoribosyltransferase.
  • the present inventors have identified loss-of-function mutations in two genes (HAAO and KYNU) of the de novo NAD synthesis pathway. Each mutation causes multiple congenital malformations including defects in the heart and vertebrae. The present inventors have also developed methods to prevent or ameliorate a symptom of the malformations.
  • congenital malformation in an unborn offspring of a subject comprising administering to the subject an effective amount of NAD or an intermediate of NAD synthesis, wherein the congenital malformation is associated with or caused by at least one of:
  • a mutation in a gene encoding an enzyme involved in NAD synthesis a dietary deficiency of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid;
  • the subject may be the mother of an unborn offspring, the unborn offspring or the born offspring.
  • the intermediate of NAD synthesis may be selected from the group consisting of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, 2- amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, 2-aminomuconate, 2- oxoadipate, glutaryl-CoA, N-methylnicotinamide (MNA), deamido-NADN-methyl-4- pyridone-3-carboxamide (4-Py), N-methyl-2-pyridone-5-carboxamide (2-Py),
  • nicotinamide mononucleotide NPN
  • nicotinic acid mononucleotide nicotinic acid adenine dinucleotide
  • deamido-NAD nicotinamide
  • nicotinamide mononucleotide nicotinamide adenine dinucleotide
  • niacin vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
  • the intermediate of NAD synthesis is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinic acid
  • the intermediate may be at least one of vitamin B3, nicotinamide or niacin (nicotinic acid).
  • the NAD or intermediate of NAD synthesis may administered at a dose of at least about 0.1 mg/kg, at least about 0.3 mg/kg, at least about 1 mg/kg, at least about 25mg/kg, at least about 50 mg/kg, at least about 100 mg/kg, at least about 150mg/kg, or at least about 200mg/kg body weight of the subject.
  • the NAD or an intermediate of NAD synthesis may be administered orally or by injection, for example by subcutaneous, intramuscular or intra-venous injection.
  • the NAD or an intermediate of NAD synthesis may be administered before conception, around the time of conception or during the term of a pregnancy, or after birth if the NAD deficiency persists.
  • the intermediate of NAD synthesis may be selected from the group consisting of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, 2- amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, 2-aminomuconate, 2- oxoadipate, glutaryl-CoA, N-methylnicotinamide (MNA), deamido-NADN-methyl-4- pyridone-3-carboxamide (4-Py), N-methyl-2-pyridone-5-carboxamide (2-Py),
  • nicotinamide mononucleotide NPN
  • nicotinic acid mononucleotide nicotinic acid adenine dinucleotide
  • deamido-NAD nicotinamide
  • nicotinamide mononucleotide nicotinamide adenine dinucleotide
  • niacin (nicotinic acid) vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
  • the intermediate of NAD synthesis may be selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide
  • the mononucleotide nicotinamide adenine dinucleotide, niacin, a derivative thereof, a salt thereof, and any combination thereof.
  • the intermediate may be at least one of nicotinamide or niacin.
  • a supplement comprising NAD or an intermediate of NAD synthesis when used for the prevention or treatment of a congenital malformation in a born or unborn offspring of a subject wherein the congenital malformation is associated with or caused by at least one of:
  • the intermediate of NAD synthesis may be selected from the group consisting of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, 2- amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, 2-aminomuconate, 2- oxoadipate, glutaryl-CoA, N-methylnicotinamide (MNA), deamido-NADN-methyl-4- pyridone-3-carboxamide (4-Py), N-methyl-2-pyridone-5-carboxamide (2-Py),
  • nicotinamide mononucleotide NPN
  • nicotinic acid mononucleotide nicotinic acid adenine dinucleotide
  • deamido-NAD nicotinamide
  • nicotinamide mononucleotide nicotinamide adenine dinucleotide
  • niacin (nicotinic acid) vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
  • the intermediate of NAD synthesis is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinic acid
  • the intermediate may be at least one of vitamin B3, nicotinamide or niacin (nicotinic acid).
  • a dose of the supplement comprises about 15mg, 18mg, 20mg, 25mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1 ,000mg, 2,000mg or about 3,000mg of NAD or an intermediate of NAD synthesis.
  • a method of detecting potential for a congenital malformation in a subject comprising:
  • the subject may be one or both parents, an unborn offspring or a born offspring.
  • the presence of the mutation is indicative of a predisposition to a congenital malformation in the subject's unborrn offspring.
  • the congenital malformation may be selected from the group consisting of a vertebral defect, sacral agenesis, spinal dysraphism, a cardiac defect, a hypoplastic kidney, a solitary kidney, a shortened long bone; a limb abnormality, submucous cleft palate, bifid uvulua; laryngeal web, vocal cord palsy, anterior anus, restrictive lung disease, microcephaly, and any combination thereof.
  • the gene may be selected from the group consisting of those listed in Table 20, namely aminoadipate aminotransferase, aminocarboxymuconate semialdehyde decarboxylase, acid phosphatase 5, arylformamidase, an alkaline phosphatase, aldehyde oxidase 1 , ADP-ribosyltransferase 1 , ADP-ribosyltransferase 2, ADP- ribosyltransferase 3, ADP-ribosyltransferase 4, ADP-ribosyltransferase 5, bone marrow stromal cell antigen 1 , catalase, CD38, cytochrome B5 reductase 3, cytochrome p450 family 2 subfamily D member 6, cytochrome p450 family 8 subfamily B member 1 , ectonucleotide pyrophosphatase/phosphodiesterase 1 , ect
  • phosphoribosyltransferase nicotinate phosphoribosyltransferase, NAD(P)HX dehydratase, NAD(P)HX epimerase, nicotinamide nucleotide adenylyltransferase 1 , nicotinamide nucleotide adenylyltransferase 2, nicotinamide nucleotide adenylyltransferase 3, nicotinamide riboside kinase 1 , nicotinamide riboside kinase 2, nicotinamide N-methyltransferase, nicotinamide nucleotide transhydrogenase, 5', 3'- nucleotidase - cytosolic, 5'-nucleotidase - cytosolic I A, 5'-nucleotidase, cytosolic IB, NT5
  • the gene may be selected from the group consisting of a gene encoding a tryptophan transporter, TD02 (tryptophan 2,3-dioxygenase), I DO 1 ,2 (Indoleamine 2,3-dioxygenase), AFMID (arylformamidase), KYNB (Kynurenine formamidase), KMO (kynurenine 3-monooxygenase), KYNU (kynureninase), HAAO (3- hydroxyanth rani late 3,4-dioxygenase), ACSMD (aminocarboxymuconate semialdehyde decarboxylase), QPRT (quinolinate phosphoribosyltransferase), NAPRT, NADSYN1 , NMAT1 , 2, or 3 (nicotinamide nucleotide adenylyltransferase 1 , 2, or 3), NADSYN1 (NA), AA, AA,
  • Riboside Kinase 1 or 2) NAMPT (nicotinamide Phosphoribosyltransferase), PNP (Purine Nucleoside Phosphorylase), PARP1-6 ( Poly(ADP-Ribose) Polymerase 1-6), ART 1-4, SIRT1-7 (Sirtuin 1-7, also known as NAD-dependent deacetylase sirtuin 1-7), CD38 (cyclic ADP ribose hydrolase), BST1 (ADP-ribosyl cyclase 2), TRPT-1 (tRNA 2- phosphotransferase 1), and any combination thereof.
  • the mutation may be in HAAO or KYNU.
  • the HAAO mutation is c.483dupT or c.559G>A.
  • the KYNU mutation is c.170-1G>T, c.1045_1051 delTTTAAGC, or c.468>A.
  • the mutation is detected by nucleic acid sequencing, multiplex ligation dependent probe amplification, single strand conformational polymorphism, or restriction fragment length polymorphism.
  • the method may further comprise administering to the subject or the subject's mother an effective amount a supplement comprising at least one intermediate of NAD synthesis according to the third aspect.
  • a method of detecting potential for a congenital malformation in a subject comprising detecting at least one of:
  • the subject may be the mother of an unborn offspring or the unborn offspring.
  • the congenital malformation is selected from the group consisting of a vertebral defect, sacral agenesis, spinal dysraphism, a cardiac defect, a hypoplastic kidney, a solitary kidney, a shortened long bone; a limb abnormality, submucous cleft palate, bifid uvulua; laryngeal web, vocal cord palsy, anterior anus, restrictive lung disease, microcephaly, and any combination thereof.
  • the method may further comprise administering to the subject or the subject's mother an effective amount a supplement comprising at least one intermediate of NAD synthesis according to third aspect.
  • vitamin B3' refers to a mixture or complex comprising nicotinic acid (niacin), nicotinamide and nicotinamide riboside.
  • 'niacin' also refers to nicotinic acid.
  • an 'intermediate of NAD synthesis' refers to any compound that is required or can be used, in the synthesis of NAD, including dietary sources such as nicotinic acid, nicotinamide, nicotinamide riboside, vitamin B3, and tryptophan.
  • Figure 1 Families with congenital malformations have HAAO or KYNU mutations and altered plasma metabolites of NAD synthesis.
  • Panel A shows pedigrees of families with congenital malformations. Symbols indicate male (square), female (circle), first trimester death/miscarriage (triangle), affected individual (patient, filled), deceased (slash). All individuals in families A, C and D were evaluated by exome sequencing. All individuals in family B were evaluated by genome sequencing. In family D two distinct variants in the KYNU gene were identified. Mutated alleles depicted as "a”, "b", “c” or “d”, "e”, reference allele (+).
  • NAD synthesis Nicotinamide adenine dinucleotide (NAD) is synthesized, and levels maintained by, three pathways: (i) the NAD de novo synthesis pathway of NAD from dietary tryptophan occurs by the kynurenine pathway; (ii) the Preiss-Handler pathway from dietary nicotinic acid; (iii) the NAD salvage pathway from nicotinamide derived from the consumption of NAD and from the diet. Tryptophan, dietary niacin (vitamin B3) supplied as nicotinic acid and nicotinamide, and nicotinamide riboside, represent dietary inputs to NAD synthesis.
  • NAD Nicotinamide adenine dinucleotide
  • NAD dinucleotide
  • NADSYN1 NAD synthase 1
  • NAm nicotinamide
  • PARP1-6 poly(ADP-ribose) polymerase
  • ART1-4 mono(ADP-ribosyl)- transferases
  • SIRT1-7 NAD+-dependent deacetylases
  • TRPT1 nicotinamide mononucleotide
  • NAMPT nicotinamide phosphoribosyltransferase
  • NMN is converted to NAD by nicotinamide nucleotide adenylyltransferases (NMNAT1-3).
  • Dietary nicotinic acid is converted to nicotinic acid mononucleotide (NaMN) by nicotinate
  • NAPRT phosphoribosyltransferase
  • Dietary nicotinamide riboside is converted to nicotinamide (NAm) by purine nucleoside phosphorylase (PNP) or to nicotinamide mononucleotide (NMN) by nicotinamide riboside kinases (NMRK1.2).
  • Nicotinamide (NAm) is methylated by nicotinamide-N-methyltransferase (NNMT) to N- methylnicotinamide (1-MNA).
  • 1-MNA is oxidized and excreted in urine (not depicted). In some instances where the gene and protein names differ, the gene name and symbol are used.
  • FIG. 3 Low levels of NAD in null mouse embryos were rescued by niacin supplement. Haao, or Kynu, homozygous null female mice were mated with heterozygous male mice. Pregnant mice were on a niacin-free diet supplemented with 5, 10 or 15 mg/L nicotinic acid in drinking water from E7.5 to 9.5. Embryos were harvested at E9.5 and total NAD(H) levels quantified. The difference between groups was tested using One way ANOVA test (all other groups vs null on 5 mg/L nicotinic acid, Dunnett multiple comparison test, based on log2 transformed data). * p ⁇ 0.05, *** p ⁇ 0.001 , **** p ⁇ 0.0001.
  • Figure 4 Verification of HAAO and KYNU mutations in affected families.
  • KYNU c.468T>A and KYNU c.1045_1051 delTTTAAGC are heterozygous in the affected proband (Family D; 11-1).
  • the father (1-1) is heterozygous for KYNU c.468T>A.
  • the mother is heterozygous for KYNU c.1045_1051 delTTTAAGC. See Figure 1 for pedigrees.
  • the variable nucleotide(s) is specified (*).
  • Figure 5 Genomic organization of HAAO and KYNU and mutation locations. Genomic organization of HAAO (upper) and KYNU (lower) and the DNA and protein variants identified in the patients. HAAO variants for patient A (black) and B (red). KYNU variants for patient C (black) and D (red).
  • FIG. 6 Mutant HAAO or KYNU proteins lack activity.
  • B HAAO overexpressing bacterial lysates were assayed for 3-hydroxyanthranilate 3,4-dioxygenase activity and the reaction rates determined.
  • FIG. 7 Generation and genotyping of Haao and Kynu mice.
  • Haao, and Kynu, null alleles were generated by CRISPR/Cas9 gene editing.
  • A Strategy for generating a null allele of Haao. Exon 2 of Haao was deleted by two flanking CRISPR guide RNAs (red arrowheads) to cause a frameshift in exon 3 (red box).
  • B Sequence and location of PAM (blue) and spacer (red) sequences relative to nucleotides of exon 2 (underlined).
  • C Strategy for generating a null allele of Kynu.
  • Exon 3 of Kynu was deleted by two flanking CRISPR guide RNAs (red arrowheads) to cause a frameshift in exon 4 (red box).
  • D Sequence and location of PAM (blue) and spacer (red) sequences relative to nucleotides of exon 3 (underlined).
  • Figure 8 Analysis of HAAO and KYNU protein expression and activity in mutant mouse lines.
  • A,E Western blot analysis of HAAO or KYNU protein from adult liver of Haao or Kynu wildtype (+/+), heterozygous null (+/-), and homozygous null (-/-) mice.
  • B, F Quantification of HAAO, or KYNU, protein by western blot.
  • C,G Enzyme reaction rate of HAAO or KYNU from adult liver of Haao or Kynu +/+, +/-, or -/- mice.
  • D, H Specific activity of HAAO and KYNU enzymes. Three mice per genotype were analyzed except Kynu +/- for which two mice were analyzed. Error bar: standard deviation
  • Figure 10 NAD-related genetic and environmental factors cause adverse pregnancy outcomes in a dose dependent manner. Miscarriage and congenital malformations are caused by recessively inherited gene mutations (dots 1-2), gene-environment interaction (dots 10-1 1), or diet alone (dots 7-8), Lines indicate that different thresholds of NE (niacin equivalent) cause miscarriage and congenital malformation with different maternal and embryo genotypes. Niacin equivalent is the amount of niacin derived from tryptophan and niacin. Mother genotype: homozygous null (dots 1 -4), heterozygous null (dots 9-12), wildtype (dots 5-8). Embryo genotype: W (wildtype); H (heterozygous null); N (homozygous null).
  • FIG 11 Miscarriage and congenital malformations can be caused by diet alone: A tryptophan-low and niacin-free diet causes embryo loss and defects in wildtype mice. Tryptophan is an essential for protein synthesis as well as NAD synthesis. To show that the defects were due to NAD deficiency and not generally due to tryptophan deficiency, the defects were prevented by adding niacin back to the diet. The incidence of viability and defects was different (p ⁇ 0.0001) between diets (Fischer's exact test). Niacin equivalent (NE) is the amount of niacin derived from tryptophan and niacin. .
  • NE Niacin equivalent
  • Figure 12 Wildtype, but not mutant HAAO and KYNU rescue loss of Bna1 and Bna5 genes in yeast.
  • a genetic complementation assay has been developed to test if variants/mutations in the human HAAO or KYNU genes function like wildtype human genes in mutant yeast. This assay can be used to test if variants in numerous human genes, that are required for NAD synthesis, alter protein function.
  • the present inventors have identified mutations that disrupt NAD synthesis and cause congenital malformations.
  • the discovery that such mutations cause congenital malformation is ground breaking and provides a basis for the use of niacin or other compounds, or intermediates of the NAD synthesis pathway to treat or prevent the malformations.
  • the invention also provides a basis for diagnostics to identify subjects that are at risk of developing the malformations.
  • the technology relates to methods of detecting defects in the de novo NAD synthesis pathway and methods of treating congenital malformations caused by defects in the de novo NAD synthesis pathway.
  • the present inventors have identified loss-of-function mutations in HAAO and KYNU genes of the de novo NAD synthesis pathway. Each mutation causes multiple congenital malformations including defects in the heart and vertebrae.
  • the present inventors have also developed methods to treat the malformations.
  • the treatment may include administration of compounds that are not formed due to the defects in the de novo NAD synthesis pathway.
  • the compound may be niacin. Congenital malformations
  • Various malformations are caused by or associated with defects in a NAD synthesis pathway such as the de novo NAD synthesis pathway or the NAD salvage pathway.
  • the defects are caused or associated with mutations in one or more genes encoding enzymes of the pathway.
  • the malformation is an irregular, anomalous, abnormal, or faulty formation, structure or anatomical feature of an embryo, fetus, infant child or adult.
  • defects in NAD synthesis may cause one or more
  • malformations selected from the group comprising:
  • vertebral defects in particular vertebral defects predominantly affecting the thoracolumbar spine
  • cardiac defects such as patent ductus arteriosus (PDA), atrial septal defect (ASD) and hypoplastic left heart (HLH);
  • limb abnormalities such as talipes (club foot);
  • laryngeal web for example with persistent laryngeal tracheomalacia
  • vocal cord palsy such as iatrogenic vocal cord palsy
  • restrictive lung disease for example restrictive lung disease due to spondylocostal defects
  • Sacral agenesis including sacral agenesis with spinal lipoma
  • Sacral tethered cord including sacral tethered cord with a terminal lipoma and/or spinal dysraphism
  • the malformation may be a neurocognitive deficit.
  • the neurocognitive deficit may be any deficit in a subject's perception, memory, association and recall in the thought process and behaviour.
  • the neurocognitive deficit may be any deficit in a subject's perception, memory, association and recall in the thought process and behaviour.
  • neurocognitive deficit may be a developmental delay, an intellectual disability, a delay in speech development or a delay in behavioural development.
  • the malformations may occur in isolation or in any combination. In some embodiments the malformations occur in combination. In one or more embodiments the malformation may be a cardiac defect, vertebral defect, limb defect, or any combination of one or more thereof. In some embodiments the same defect in AD synthesis will cause different malformations or combination of malformations in different subjects.
  • malformation is used to refer to the consequence of a physical or morphological malformation or neurocognitive deficit such as those set out above.
  • a malformation may be miscarriage for example a pregnant female may be gestating an embryo with a malformation.
  • the gene may be selected from the group consisting of any gene listed in Table 20, that is the gene may be selected from the group consisting of aminoadipate aminotransferase, aminocarboxymuconate semialdehyde decarboxylase, acid phosphatase 5, arylformamidase, an alkaline phosphatase, aldehyde oxidase 1 , ADP- ribosyltransferase 1 , ADP-ribosyltransferase 2, ADP-ribosyltransferase 3, ADP- ribosyltransferase 4, ADP-ribosyltransferase 5, bone marrow stromal cell antigen 1 , catalase, CD38, cytochrome B5 reductase 3, cytochrome p450 family 2 subfamily D member 6, cytochrome p450 family 8 subfamily B member 1 , ecto nucleotide
  • adenylyltransferase 3 nicotinamide riboside kinase 1 , nicotinamide riboside kinase 2, nicotinamide N-methyltransferase, nicotinamide nucleotide transhydrogenase, 5', 3'- nucleotidase - cytosolic, 5'-nucleotidase - cytosolic I A, 5'-nucleotidase, cytosolic IB, NT5C1 B-RDH14 readthrough, 5'-nucleotidase - cytosolic II, 5'-nucleotidase - cytosolic IIIA, 5'-nucleotidase - cytosolic NI B, 5'-nucleotidase ecto, 5',3'-nucleotidase - mitochondrial, nudix hydrolase 12, poly(
  • TD02 tryptophan 2,3-dioxygenase
  • IDO 1 ,2 Indoleamine 2,3-dioxygenase
  • AFMID arylformamidase
  • KYNB Kynurenine formamidase
  • KMO kynurenine 3-monooxygenase
  • NAPRT NAPRT
  • NADSYN1 NMAT1 , 2, or 3
  • NADSYN1 NAD synthetase 1
  • nicotinamide phosphoribosyltransferase NAPRT
  • NADSYN1 NMAT1 , 2, or 3
  • NADSYN1 NAD synthetase 1
  • nicotinamide phosphoribosyltransferase NAPRT
  • NADSYN1 NMAT1 , 2, or 3
  • NADSYN1 NAD synthetase 1
  • NAD synthetase 1 nicotinamide phosphoribosyltransferase
  • NMRK1.2 Neurotinamide Riboside Kinase 1 or 2
  • NAMPT nicotinamide Phosphoribosyltransferase
  • PNP Purine Nucleoside Phosphorylase
  • PARP1-6 Poly(ADP-Ribose) Polymerase 1-6
  • ART 1-4 SIRT1-7 (Sirtuin 1-7, also known as NAD-dependent deacetylase sirtuin 1-7)
  • CD38 cyclic ADP ribose hydrolase
  • BST1 ADP-ribosyl cyclase 2
  • TRPT-1 tRNA 2-phosphotransferase 1
  • the gene is HAAO or KYNU.
  • the types of mutations that lead to defects in a NAD synthesis pathway include a point mutation such as a missense mutation, nonsense mutation or a synonymous mutation, a duplication, a deletion, an insertion or a splice mutation.
  • the mutation may be homozygous or heterozygous.
  • the malformation may not be as severe as the corresponding homozygous mutation.
  • a subject with a heterozygous mutation may exhibit fewer malformations or a different combination of malformations than a subject with the corresponding heterozygous malformation.
  • a subject with a heterozygous mutation may exhibit only a single malformation.
  • Subjects with a heterozygous mutation may exhibit reduced severity of a malformation compared to a subject with a corresponding homozygous mutation.
  • mutations can be detected using fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), PCR amplification of a gene or portion thereof and sequencing of the amplification product; sequencing such as whole-genome sequencing (WGS) or whole exome sequencing (WES), use of microarrays, multiplex ligation-dependent probe amplification (MLPA), single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis or restriction fragment length polymorphism (RFLP).
  • Nucleic acid for mutation detection can be obtained from samples of blood, skin, amniotic fluid, chorionic villi.
  • defects in a NAD synthesis pathway can be detected by measuring the concentrations of metabolites produced by the enzymes of the pathway, either quantitatively or qualitatively.
  • a defect a NAD synthesis pathway can be identified by measuring the level of NAD.
  • the metabolites may be selected from the group comprising tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HAA), xanthurenic acid, quinolinic acid (also known as quinolinate), 2-aminomuconate, 2-oxoadipate, glutaryl-CoA, nicotinic acid mononucleotide, nicotinic acid adenine
  • NAD dinucleotide
  • NAD deamido-NAD
  • NAD nicotinamide
  • MNA N-methylnicotinamide
  • 4-Py deamido-NADN-methyl-4-pyridone-3- carboxamide
  • 2-Py N-methyl-2-pyridone-5-carboxamide
  • NMN nicotinamide mononucleotide
  • a stable product derived therefrom e.g. cinnabarinic acid
  • the metabolite may be anthranilate, kynurenate, xanthurenate or any combination thereof.
  • NAD and metabolites can be detected, quantified or both using mass spectrometry, typically in conjunction with liquid chromatography.
  • mass spectrometry typically in conjunction with liquid chromatography.
  • 3-hydroxykynurenine and 3-hydroxyanthranilic acid can be detected and quantified by liquid chromatography-tandem mass spectrometry (LC- MS/MS).
  • LC- MS/MS liquid chromatography-tandem mass spectrometry
  • a serum sample is deproteinated with trichloroacetic acid before neutralisation. The sample is then applied to a C18 column and metabolites eluted using as gradient of 0-5 % mobile phase (e.g. acetic acid in acetonitrile).
  • metabolite's are then applied to a mass spectrometer for example a triple quadrupole mass spectrometer to detect 3HK and 3HAA by multiple reaction monitoring (MRM) in positive ion mode.
  • MRM multiple reaction monitoring
  • the metabolite's can be quantified against commercial standards for example those available from Sigma Aldrich (USA).
  • the measured concentration of NAD or a metabolite of the pathway can be compared to the concentration of NAD or a metabolite in a normal individual.
  • NAD or metabolites can be measured in samples of blood, serum, plasma, saliva, urine, tear, lymph fluid, cerebrospinal fluid, mucosal secretion, peritoneal fluid, ascitic fluid, fecal matter, amniotic fluid, chorionic villus and body exudate.
  • defects in NAD synthesis of a subject in utero can be detected by measuring the concentrations of metabolites in a maternal sample.
  • a sample of blood, serum, plasma, saliva, or urine may be obtained from a pregnant female for analysis.
  • NAD or metabolites of NAD synthesis or nicotinamide degradation are measured in a blood or urine sample.
  • a defect in NAD synthesis is detected, for example by measuring metabolite levels or detecting a gene mutation, in a subject that is considering becoming pregnant. In this way the methods described herein can be used as prepregnancy testing for a defect in NAD synthesis.
  • a subject may have a heterozygous mutation in a gene encoding an enzyme involved in NAD synthesis.
  • a malformation may not be apparent or the severity of a symptom associated with a malformation may be minimal.
  • heterozygous mutations may produce sufficient enzymes associated with NAD synthesis that sufficient NAD is produced at certain times, for example when the dietary intake of tryptophan, vitamin B6 (pyridoxal phosphate) or niacin is sufficient.
  • vitamin B6 pyridoxal phosphate
  • niacin a digestive disorder that prevents the uptake of, for example vitamin B6, niacin or tryptophan such as often occurs during pregnancy
  • the subject may be at risk of for example increasing the severity of a symptom associated with a malformation. This is particularly the case for a subject's offspring when the offspring is in utero.
  • a subject with a heterozygous mutation in a gene encoding an enzyme involved in NAD synthesis may normally produce sufficient NAD, however, in times of physiological stress such as restriction of vitamin B6, niacin or tryptophan that may occur during pregnancy, the offspring, who may also have a heterozygous mutation may be at greater risk of developing a malformation.
  • a subject with a heterozygous mutation in a gene encoding an enzyme involved in NAD synthesis is at greater risk of producing an offspring with a malformation if both of the offspring's biological parents have a heterozygous mutation in a gene encoding an enzyme involved in NAD synthesis.
  • the risk of a subject's offspring having a congenital malformation caused by a defect in NAD synthesis can be determined by assessing whether one or more of the offspring's biological parents have a defect in NAD synthesis.
  • a sample from one of the biological parents can be tested for a mutation in a gene encoding an enzyme involved in NAD synthesis. If a mutation is found, and is heterozygous then this is indicative of a predisposition to a congenital malformation in the subject's offspring.
  • a sample from one of the biological parents can be tested the concentration of NAD, a metabolite of NAD synthesis or degradation, or a stable product from a metabolite in the sample. That concentration can be compared to the concentration of NAD, the metabolite or the stable product to a predetermined concentration of NAD, the metabolite or the stable product in a normal subject (i.e. one without a defect in NAD synthesis). If the concentration of NAD, the metabolite or the stable product is lower than that of a normal subject then that is indicative of a predisposition to a congenital malformation in the subject's offspring.
  • the risk of a subject's offspring having a congenital malformation caused by a defect in NAD synthesis can be determined before conception of the offspring, for example by detecting defects in NAD synthesis using any of the methods disclosed herein.
  • compounds useful for the treatment of congenital malformations caused by defects in NAD pathway are compounds, or salts or derivatives thereof, that are intermediates in the pathway downstream of the defect or the end product of the pathway, NAD or substrates and metabolites of the salvage pathway of NAD synthesis.
  • Compounds useful for the treatment of a malformation include the following: tryptophan, N-formylkynurenine, kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, nicotinic
  • nicotinic acid mononucleotide nicotinic acid adenine dinucleotide (or deamido- NAD), nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin (nicotinic acid), vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
  • Useful compounds for the detection of a defect in NAD synthesis include of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3- hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HAA), xanthurenic acid, quinolinic acid (also known as quinolinate), 2-aminomuconate, 2-oxoadipate, glutaryl-CoA, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide (NaAD)(or deamido- NAD), nicotinamide adenine dinucleotide (NAD), nicotinamide, N-methylnicotinamide (MNA), deamido-NADN-methyl-4-pyridone-3-carboxamide (4-Py), N-methyl-2-pyridone- 5-carboxamide (2-Py),
  • quinolinic acid niacin
  • nicotinamide adenine dinucleotide nicotinamide and nicotinamide mononucleotide.
  • the malformation may be treated by tryptophan, 3- hydroxyanth rani lie acid, 2-amino-3-carboxymuconic semialdehyde; quinolinic acid, niacin, nicotinamide adenine dinucleotide, nicotinamide, nicotinamide mononucleotide, derivatives thereof, salts thereof, and any combination thereof.
  • niacin is used to treat the malformation.
  • a compound described herein can be prepared as a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to a derivative of the disclosed compounds where the parent compound is modified by making an acid or base salt.
  • Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.
  • conventional nontoxic salts include those derived from bases, such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like.
  • salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985).
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
  • a compound described herein can be formulated in combination with one or more other compounds including another compound described herein.
  • a compound described herein can be formulated as a pharmaceutical composition or as a dietary supplement and administered to a mammalian host, such as a human patient or non-human animal, in a variety of forms adapted to the chosen route of administration.
  • a composition or supplement sometimes includes a diluent, buffer, preservative and the like.
  • Various sustained release systems for drugs have also been devised, and can be applied to the compounds described herein.
  • compositions described herein may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the subject's diet.
  • an active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, or wafers.
  • Such compositions and preparations sometimes contain at least 0.1 % of active compound.
  • the percentage of the compositions and preparations may be varied and sometimes are about 2% to about 60% of the weight of a given unit dosage form.
  • the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • Tablets, troches, pills, and capsules may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
  • a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • the active compound may be incorporated into sustained-release preparations and devices.
  • a compound may be administered by infusion or injection (for example subcutaneous, intramuscular or intravenous injection).
  • Solutions of a compound or a pharmaceutically acceptable salt thereof can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof. These preparations sometimes contain a preservative to prevent the growth of microorganisms.
  • a dosage form can include a sterile aqueous solution or dispersion or sterile powder comprising an active ingredient, which are adapted for the preparation of sterile solutions or dispersions.
  • the ultimate dosage form can be a sterile fluid which is stable under the conditions of manufacture and storage.
  • a liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), nontoxic glyceryl esters, and suitable mixtures thereof.
  • microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • An isotonic agent for example, a sugar, buffer or sodium chloride is included in some embodiments.
  • Prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile solutions often are prepared by incorporating an active compound in a required amount in an appropriate solvent, sometimes with one or more of the other ingredients enumerated above, followed by filter sterilization.
  • preparation methods sometimes utilized are vacuum drying and the freeze drying techniques, which yield a powder of an active ingredient in addition to any additional desired ingredient present in the previously sterile-filtered solutions.
  • niacin niacin, nicotinic acid and nicotinamide are approved for therapeutic use in humans for treating niacin deficiency.
  • RDA recommend daily allowance
  • niacin 16 milligrams per day for men and 14 milligrams per day for women.
  • Conventional medical advice indicates that excess niacin or nicotinic acid can be harmful and may adversely affect liver function.
  • doses of 50mg/kg of niacin for example are useful in the treatment of malformations caused by defects in NAD synthesis.
  • the compound such as niacin
  • a controlled release tablet may comprise about 5-30% high viscosity hydroxypropyl methyl cellulose, about 2-15% of a water- soluble pharmaceutical binder, about 2-20% of a hydrophobic component such as a waxy material, e.g., a fatty acid, and about 30-90% niacin.
  • a suitable controlled release tablet comprises: (a) about 5-20 percent by weight hydroxypropyl methylcellulose having a viscosity of about 10,000 CPS or greater, a substitution rate for the methoxyl group of about 7-30% and a substitution rate for the hydroxypropoxyl group of about 7-20%; (b) about 2-8 percent hydroxypropyl
  • methylcellulose having a viscosity of less than about 100, CPS methyl cellulose, or polyvinyl pyrollidone; (c) about 5-15 percent by weight hydrogenated vegetable oil or stearic acid; and (d) about 30-90% niacin.
  • the controlled released tablets can also be coated so as to further prolong the release of the niacin into the gastrointestinal tract, or to prevent its release into the stomach, in order to prevent or attenuate the gastrointestinal side effects which can accompany niacin administration.
  • coatings predominantly comprising a polymeric material having a high degree of swelling on contact with water or other aqueous liquids can be used to further prolong the release of the compound from the tablet.
  • suitable polymers include cross-linked sodium carboxymethylcellulose, cross-linked
  • hydroxypropylcellulose hydroxymethylpropylcellulose, e.g., Methocel®,
  • carboxymethylamide potassium methylacrylate divinylbenzene copolymer, polymethyl methacrylate, cross-linked polyvinylpyrrolidine, high molecular weight polyvinylalcohol, and the like.
  • Hydroxypropylmethyl cellulose is available in a variety of molecular weights and viscosity grades for example from Dow Chemical Co. under the Methocel® brand name. These polymers may be dissolved in suitable volatile solvents, along with dyes, lubricants, and flavorings, and coated onto the controlled release tablets, e.g., in amounts equal to 0.1-5% of the total tablet weight, by methods well known to the art. For example, see Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Publishing Co., Easton, Pa. (16th ed. 1980).
  • the controlled release tablets for example niacin tablets, can be formulated to contain about 5mg, 10mg, 15mg, 20, mg, 30mg, 40mg, 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 825mg, 850mg, 875mg, 900mg, 925mg, 950mg, 975mg or about 1 ,000 mg, 1 ,250mg, 1 ,500mg, 1 ,750mg, 2,000
  • the controlled release tablets will release about 10-35 wt-% of the total niacin within about 2 hours in an in vitro dissolution test, and about 40- 70 wt-% of the total niacin in eight hours.
  • the concentration of a compound described herein in a liquid composition is about 0.1 wt %, 0.25wt %, 0.5wt %, 0.75wt %, 1wt %, 2wt %, 3wt %, 4wt %, 5wt %, 6wt %, 7wt %, 8wt %, 9wt %, 10wt %, 1 1wt %, 12wt %, 13wt %, 14wt %, 15wt %, 16wt %, 17wt %, 18wt %, 19wt %, 20wt %, 21wt %, 22wt %, 23wt %, 24wt %, 25wt %, 26wt %, 27wt %, 28wt %, 29wt %, 30wt %, 31wt %, 32wt %, 33wt %, 34w
  • Useful dosages, whether by controlled release or not, of compounds can be determined by their in vivo activity in animal models, such as the mouse models described herein. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art. Treatment of Congenital Malformations
  • compositions and formulation disclosed herein are useful for the treatment or prevention of malformations in a subject associated with or caused by defects in NAD synthesis.
  • the defects may be due to mutations in one or more genes encoding enzymes active in NAD synthesis such as the genes listed in Table 20 which include genes encoding a tryptophan transporter, TD02 (tryptophan 2,3- dioxygenase), IDO 1 ,2 (Indoleamine 2,3-dioxygenase), AFMID (arylformamidase), KYNB (Kynurenine formamidase), KMO (kynurenine 3-monooxygenase), KYNU
  • NAPRT NAPRT
  • NADSYN1 NMAT1 , 2, or 3
  • NADSYN1 NAD synthetase 1
  • nicotinamide phosphoribosyltransferase NAPRT
  • NADSYN1 NMAT1 , 2, or 3
  • NADSYN1 NAD synthetase 1
  • nicotinamide phosphoribosyltransferase NAPRT
  • NADSYN1 NMAT1 , 2, or 3
  • NADSYN1 NAD synthetase 1
  • NAD synthetase 1 nicotinamide phosphoribosyltransferase
  • NMRK1 ,2 Natural Organotase 1 or 2
  • NAMPT nicotinamide Phosphoribosyltransferase
  • PNP Purine Nucleoside Phosphorylase
  • PARP1-6 Poly(ADP-Ribose) Polymerase 1-6
  • ART 1-4 SIRT1-7 (Sirtuin 1-7, also known as NAD-dependent deacetylase sirtuin 1-7)
  • CD38 cyclic ADP ribose hydrolase
  • BST1 ADP-ribosyl cyclase 2
  • TRPT-1 tRNA 2-phosphotransferase 1
  • the malformations may be for example cardiac malformations, vertebral, renal dysplasia, limb anomalies, and tracheoesophageal fistula, for example
  • TE esophageal atresia
  • terapéuticaally effective amount refers to an amount of a compound provided herein, or an amount of a combination of compounds provided herein, to treat or prevent a malformation, or to treat a symptom of the malformation, in a subject.
  • the terms 'subject' and 'patient' are used interchangeably to refers to an individual who will receive or who has received treatment (e.g.,
  • a compound described herein may be in a therapeutically effective amount in a formulation or medicament, which is an amount that can lead to a physiological effect (e.g., inhibiting the malformation), or lead to ameliorating, alleviating, lessening, relieving, diminishing or removing symptoms of the malformation, for example.
  • a compound described herein can be administered to any subject having a malformation caused by a defect in NAD synthesis. Examples of a subject include mammal, human, ape, monkey, ungulate (e.g., equine, bovine, caprine, ovine, porcine, buffalo, camel), canine, feline, rodent (e.g. mouse, rat).
  • a subject may be male or female.
  • the compound can be administered to a subject in any age group, including, for example, neonate, infant, juvenile, paediatric, adolescent and adult. In some
  • the compound for example where the subject is in utero the compound can be
  • a compound described herein can be administered to any subject to reduce the risk of a malformation caused by a defect in NAD synthesis.
  • a compound described herein can be administered to any subject to increase the amount of NAD in the subject to reduce the risk of a malformation caused by a defect in a NAD synthesis pathway, or to prevent such a malformation becoming apparent.
  • a compound described herein can be administered to a subject who is, or is contemplating becoming pregnant in order to reduce the risk of a
  • a compound described herein can be administered to a subject who is, or is contemplating becoming pregnant in order to reduce the risk of miscarriage, for example miscarriage caused by a malformation.
  • the methods involve administering a therapeutically effective amount of a compound to the subject with the malformation.
  • the compound may be administered by any route,
  • the compound may be administered by oral, parenteral, intravenous, intramuscular, topical, subcutaneous or intradermal routes.
  • the route of administration will vary depending on the nature of the compound and the way it is formulated. A skilled person will be able to determine the appropriate route of administration.
  • the subject is in utero and administration of the compound to the subject can be achieved by administering the compound to the mother of the subject.
  • the compound is administered by injection, for example a subcutaneous, intramuscular or intravenous injections.
  • the compound is administered orally.
  • a suitable dose is in the range of from about 0.1 mg to about 200 mg/kg, e.g., from about 50 to about 125 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, and often is in the range of 6 to 90 mg/kg/day, or about 15 to 60 mg/kg/day.
  • a suitable dose in general, sometimes is in the range of from about 1 to 150 mg/kg body weight of the recipient per day, e.g. from about 10 to about 130 mg/kg, from about 40 to about 120 mg/kg, from about 50 to about 100 mg/kg, from about 60 to 90 mg/kg, from about 65 to 85 mg/kg, or, for example, about 80 mg/kg/day.
  • the compound may be administered at a dose of at least about 50mg/kg body weight of the subject.
  • the compound may be administered at a dose of about 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45,46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93,
  • a compound may be conveniently administered in unit dosage form.
  • a desired amount of compound may be administered in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four, five, six, seven, eight, nine, ten or more than ten doses per day.
  • a suitable dose is significantly more than the RDA for that compound.
  • the dose may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, or 170 or more times the RDA for that compound.
  • Example 1 Study Participants
  • Family A Patient A was the male twin of a dizygotic pregnancy to first cousins once removed from Iraq. The mother had a previous miscarriage at 12 weeks gestation, with possible spinal defects. The other four children in the family are healthy.
  • Family B Patient B was the female twin of a dizygotic pregnancy, born at 38 weeks gestation, to first cousins from Riverside. Her male twin and two older brothers are well and there is no family history of congenital heart disease, congenital anomalies or developmental delay. Her mother had gestational diabetes requiring insulin in the second half of the pregnancy.
  • Patient B was antenatally diagnosed with hypoplastic left heart (HLH). She was born at term with a birth weight of 2680 g (13th percentile), 270 g lighter than her male twin, and OFC 31.2cm (1st percentile). Apgar scores were 9 and 9 at one and five minutes respectively. Chromosome microarray detected no malformations. Postnatal echocardiogram confirmed the HLH with mitral stenosis, aortic stenosis and a
  • hypoplastic aortic arch In the first week of life she underwent the Norwood procedure with the Sano shunt modification. Spinal imaging showed multiple thoracolumbar segmentation defects with abnormal rib configuration and butterfly vertebrae and a hypoplastic distal sacrum with sacral spinal dysraphism, tethered cord and terminal lipoma. She had a small dysplastic right kidney with normal renal function. She had left- sided moderate sensorineural hearing loss, no imaging of the auditory system was performed. She had a weak cry and dysphagia due to left vocal cord palsy, likely due to intraoperative damage of the recurrent laryngeal nerve. No laryngeal structural anomalies were detected endoscopically. At age 12 weeks, she was assessed as high risk for future cerebral palsy and movement problems, and early intervention was recommended. At 1 1 months of age, she died from complications of HLH.
  • Echocardiogram demonstrated a patent ductus arteriosus (PDA) that worsened with time, and led to increased requirements for respiratory support; surgical intervention was not performed.
  • Abdominal ultrasound revealed small kidneys.
  • the baby had no radial ray defects but had mild rhizomelic shortening, right talipes equinovarus, and bilateral 2-3 toe syndactyly. She had low-set ears, a small anterior forehead, upslanting small palpebral fissures and a short neck.
  • the baby died at 4 months of age of restrictive lung disease due to thoracic spondylocostal defects.
  • Family D Patient D was the first live-born child to non-consanguineous North American parents. She was preceded by two first trimester losses. She has a healthy younger sister. There was no known teratogen exposure during the pregnancy, but the mother had a BMI of 29.3 kg/m 2 .
  • Table 1 summarizes patient clinical features, the identified DNA and protein variants, and fold changes in the levels of upstream (3HAA, 3HK) and downstream (NAD) metabolites in patient plasma compared with unaffected heterozygous family members.
  • ASD atrial septal defect
  • HLH hyperplastic left heart
  • PDA patent ductus arteriosus
  • VUR vesicoureteric reflux
  • SNHL sensorineural hearing loss
  • GDD global developmental delay
  • ID integer disability
  • HAAO 3- hydroxyanth rani late 3,4-dioxygenase
  • KYNU kynureninase
  • 3HAA 3- hydroxyanth rani lie acid
  • NAD nicotinamide adenine dinucleotide
  • na not available
  • “+" denotes present
  • -" denotes absent.
  • NAD(H) is the sum of NAD+ and NADH. See Figures 2-5, Table 2, Table 12.
  • Family B (individuals 1.1 , 1.2, II.3, II.4): DNA sequencing libraries were made using the TruSeq Nano DNA HT Sample Prep Kit (lllumina Inc., California, CA, USA). Following clustering of each library on a single lane of a V2.5 patterned flowcell, paired- end sequencing with 150 bp read length was performed using the lllumina HiSeq X, (Garvan Institute, Australia).
  • VCF Variant Call Format
  • Each family's ANNOVAR-annotated variant file was viewed using the VarSifter application. Variants were retained when reads were present for that site in all family members. Potential disease-causing variants were identified following a series of filtering steps based on: the predicted mode of disease inheritance; the variant's effect on the gene (non-synonymous); minor allele frequency using the variant frequencies of multiple control databases (1000 Genomes, ExAC, ESP6500) and pathogenicity predictions of multiple bioinformatics algorithms (i.e. Polyphen-2,8 SIFT, MutationTaster. Manual verification of variant calls was performed by visual inspection of the sequence in question using the Integrative Genomics Viewer (IGV).
  • IGF Integrative Genomics Viewer
  • HAAO 3-Hydroxyanthranilate 3,4-dioxygenase
  • Kynureninase (KYNU) catalyzes the conversion of 3-hydroxykynurenine (3HK) to 3HAA ( Figure 2).
  • the human HAAO cDNA clone was purchased from Mammalian Gene Collection (Accession No: BC029510).
  • the human KYNU cDNA clone was purchased from Origene (RC214932). Using these plasmids as templates, site-directed
  • mutagenesis was carried out to introduce specific mutations to the HAAO and KYNU cDNA using the KAPA HiFi PCR Kit (Kapa Biosystems) and appropriate mutagenic oligonucleotides.
  • the wildtype and mutated HAAO and KYNU open reading frame were then cloned into pT7 MAT-Tag® FLAG®-1 Expression Vector (Sigma-Aldrich) at the Hindi 11 and EcoRI sites.
  • the expression constructs were sequenced to confirm that no secondary mutations arose from PCR amplification.
  • the expression constructs were transformed into the E. coli BL21 (DE3) strain. Bacteria were grown in 0.5 L LB media containing 100 ⁇ g/mL ampicillin at 37°C until OD600 reached 0.6. Protein expression was induced by adding 0.5 mM IPTG and the cultures were incubated at 16 °C for a further 16h. Bacteria were pelleted, resuspended in lysis buffer (100 mM Tris-HCI pH 8, 0.3 M NaCI, 20 mM imidazole, 1x completeTM, EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM PMSF, 0.05% Tween 20) and lysed by sonication on ice for 50 sec.
  • lysis buffer 100 mM Tris-HCI pH 8, 0.3 M NaCI, 20 mM imidazole, 1x completeTM, EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM PMSF, 0.05% Tween 20
  • NP-40 0.5% NP-40 was included in the lysis buffer for the bacteria expressing the HAAO protein.
  • the lysate was centrifuged at 21 ,000 rcf for 15 min at 4°C and the supernatant was passed through a 0.22 ⁇ m filter.
  • the his-tagged KYNU recombinant proteins were purified by immobilized metal ion affinity chromatography using the AKTApurifier liquid chromatography system (GE Healthcare) and a 1 ml_ HisTrap HP column (GE Healthcare). Bound protein was eluted with elution buffer containing 0.3 M NaCI, 100 mM Tris-HCI pH 8, 0.05% Tween 20, and a step-wise increasing concentration of imidazole from 20 mM to 250 mM.
  • Peak fractions were analyzed by SDS-PAGE to determine the fraction that contained the highest amount of recombinant protein.
  • concentration of the recombinant protein was estimated on the Coomassie stained SDS-PAGE gel by quantifying the intensity of the specific bands against known amounts of BSA as a standard.
  • concentrations of the HAAO proteins in the bacterial crude lysates were estimated by western blotting against FI_AG-tag (1 : 1000, F1804, Sigma Aldrich) using known amounts of recombinant HAAO protein as a standard.
  • HAAO 3-Hydroxyanthranilate 3,4-dioxygenase activity was measured spectrophotometrically according to Walsh et al (1991 ).1 1 Briefly, 7 ⁇ L of bacterial lysates were loaded into a 96-well plate in duplicate wells. The reaction was initiated at room temperature by adding 200 ⁇ L of reaction mixture containing 0.3 mM
  • Kynureninase (KYNU) activity was measured fluorometrically in a 96-well plate as described by (Gaertner et a/ J Bacteriol 1971 ; 108(2): 902-9). Briefly, 50 ⁇ L of purified recombinant KYNU was added into a 96-well plate in duplicate wells. The reaction was initiated at room temperature by adding 200 ⁇ L of reaction mixture containing 4 ⁇ pyridoxal phosphate, 0.1 mM 3-hydroxy-DL-kynurenine (3HK) in 10 mM Tris-HCI pH 8.
  • the reaction was monitored fluorometrically every 5 sec for 1 min with a FLUOstar plate reader (BMG Labtech) using the excitation and emission wavelengths of 340 nm and 405 nm, respectively.
  • the specific activity of enzyme was defined as the amount of 3- hydroxyanth rani lie acid (3HAA) produced per mg of enzyme per min, using known amounts of 3HAA as a standard.
  • Metabolites were quantified in human plasma using high-performance liquid chromatography and gas chromatography-mass spectrometry as described in the Supplementary Appendix. Metabolites were quantified in mouse serum using liquid chromatography-tandem mass spectrometry as described in the Supplementary
  • Serum and tissue NAD were measured using the NAD/NADH-GloTM Assay kit (Promega).
  • mice were fed a complete diet (90 mg/kg niacin and 3.7 g/kg tryptophan) (Gordons Premium Breeder Rat & Mouse, Gordons Specialty Feeds, Bargo, Australia), or a niacin-free diet (NFD; 1.8 g/kg tryptophan) (SF16-049, Specialty Feeds, Glen Forrest, Australia) with 0, 5, 10 or 15 mg/L nicotinic acid in drinking water. Embryos were harvested at various stages and analyzed as described below.
  • CRISPR guide RNAs were designed using CRISPR Design tool
  • Haao Guide RNAs were designed with spacers targeting either side of exon 2 (5'-TGGTGACACGTTAATGCGTG-3 ⁇ 5'-CACTTCCTAG ACG AGTCCTA-3') . Founder pups were screened for exon deletion by PCR amplification across the targeted region (F-5'-GCCCAAGATTGCAATAAAGC-3 ⁇ R-5' -GTTCCTTTCCGTCCTCCATT-3') .
  • Kynu Guide RNAs were designed with spacers targeting either side of exon 3 (5'-GGCATAACTTAAACCCACAG-3 ⁇ 5'-AAGTTACACCCTAGCATATT-3') . Founder pups were screened for exon deletion by PCR amplification across the targeted region (F-5'-GAAAGAGCAAAAGATCTAGAGACCA-3 ⁇ R-5' -CATCAGTGTCACCCAGCCTA- 3').
  • mutant mice we used CRISPR/Cas9 genome editing to induce double stranded breaks flanking exon 2 of Haao, or exon 3 of Kynu- 001, with a view to create a frameshift mutation early in the open reading frame to disrupt protein function. Eleven founder mice were generated for Haao that deleted exon 2 as judged by PCR across the target exon. Five of these lines had a frameshift mutation confirmed by Sanger sequencing.
  • Haao founder 4 was chosen for further analysis; it carries an allele with a 325 base pair deletion (c.82-167_161-4232del/p.His28Cysfs*8) that encodes for a truncated protein consisting of 28 N-terminal amino acids of HAAO. Eight founder mice were generated for Kynu that deleted exon 3 as judged by PCR across the target exon. Four of these lines had a frameshift mutation demonstrated by Sanger sequencing.
  • Kynu founder 20 was chosen for further analysis; it carries an allele with a 409 base pair deletion (c.253-200_294-7827del/p.lso58Argfs*7) that encodes for a truncated protein consisting of 58 N-terminal amino acids of KYNU.
  • mice and embryos were genotyped by PCR. DNA samples were prepared from tails, yolk sacs, or whole embryos as described previously (Sasaki et al. Journal of Biological Chemistry 2015;290(28): 17228-38). All mouse analyses were conducted by individuals blinded to their genotype.
  • Heterozygous null mice were intercrossed and liver harvested from the 6-8 weeks old offspring.
  • the livers were lysed in 1x LDS sample buffer (Thermo Fisher Scientific) and subject to western blot analysis using the primary antibodies anti-Haao (1 : 1500, NBP1-77361 , Novus Biologicals), anti-Kynu (1 :500, 11796-1-AP, Proteintech) and anti-tubulin (1 :500, E3, Developmental Studies Hybridoma Bank) and the secondary antibodies Goat anti-rabbit AlexaFluor® 680 (1 :5000, Life Technologies) and Goat anti- mouse IRDye® 800 (1 :5000, LI-COR).
  • the clinical features of the study participants are summarized in Figure 1 and Table 4.
  • the four families include one consanguineous family from Iraq (Family A), two consanguineous families from Lebanon (Families B and C), and a family from North America (Family D) ( Figure 1).
  • the mother in Family B had insulin-requiring gestational diabetes.
  • the mother in Family C had pre-pregnancy insulin-requiring diabetes, hypercholesterolemia, and a body mass index (BMI) in the obese range.
  • the mother in Family D had a BMI borderline obese.
  • Patients A and B were each a dizygotic twin.
  • Patient A had a submucous cleft palate, bifid uvula, and a laryngeal web with persistent laryngeal tracheomalacia.
  • Patient B had left vocal cord palsy that was possibly iatrogenic. None of the patients had a tracheoesophageal defect.
  • Patient C had an anterior anus. Patient C died at age 4 months of restrictive lung disease due to spondylocostal defects and Patient B at age 11 months due to HLH complications. In addition to congenital malformations, postnatal growth and cognitive defects were evident.
  • Patients A to C were microcephalic.
  • Patients A and D have extreme short stature. Patient A has moderate intellectual disability, and behavioral issues at age 12 years, and Patient D has speech delay at age 3 years.
  • Example 7 Identification of HA AO and KYNU Mutations in Patients with Multiple Congenital Defects
  • Genomic sequencing was used to identify disease-causing variants in coding exons and splice sites.
  • variants were filtered according to a recessive inheritance model.
  • variants were also filtered according to a compound heterozygous inheritance model, or for a de novo mutation in the patient. Additional filtering selected variants that were non-synonymous, rare, and predicted to be damaging, and these variants were assessed for further evidence of disease causation (Tables 5-9).
  • Variants that introduced a premature stop codon (stop-gain) or disrupted transcript splicing were considered more likely to be damaging than missense variants.
  • Family B the patient was homozygous for a c.558G>A variant in HAAO leading to a stop codon (p.W186*). The parents and unaffected siblings were heterozygous for this variant.
  • Family C the patient was homozygous for a c.170- 1G>T splicing variant in KYNU leading to a stop codon downstream (p.V57Efs*21). The parents were heterozygous for this variant and the unaffected siblings were either heterozygous or homozygous reference.
  • the identified stop-gain variants are predicted to lead to nonsense-mediated decay of the transcripts. In case this does not occur in vivo, we tested the activity of the truncated enzymes. For HAAO the conversion of 3HAA to ACMS was quantified and for KYNU the conversion of 3HK to 3HAA. All the identified HAAO and KYNU mutants, as well as KYNU p.T198A that is associated with hydroxykynureninuria but not congenital malformation were tested. The specific activity of both the truncated HAAO and KYNU enzymes was either completely lost or greatly reduced compared with wildtype ( Figure 6, Table 10). By contrast, the activity of KYNU p.T198A, had 64% of wildtype enzyme activity.
  • the inter- and intra-assay coefficient of variation is within the acceptable range of 3-7%.
  • PA and QA were concurrently detected using an Agilent 7890 gas chromatograph coupled with an Agilent 5975 mass spectrometer in accordance to method previously described.16.
  • 50 ⁇ L of extracted samples containing deuterated internal standards were derivatized with trifluoroacetic anhydride and hexafluoroisopropanol for an hour at 60°C.
  • the derivatized compound is then dissolved in toluene with 1 ⁇ L of this solution injected onto a DB-5MS capillary column (Agilent Technologies, Inc, Santa Clara, CA). Analysis was carried out with mass spectrometer operating in electron capture negative ionization mode and simultaneously monitored for selected ions (m/z 273 for PA, m/z 277 for 2H4-PA, m/z 467 for QA and m/z 470 for 2H3-QA).
  • Concentration of PA and QA are interpolated from the established six-point calibration curve based on abundance count ratio of the metabolites to their corresponding deuterated internal standards within each samples.
  • the inter- and intra-assay CV met a 7-10% acceptability criterion.
  • Example 10 Mice Null for HA AO, or KYNU, Model the Disease when NAD is Limiting
  • mice Intercrosses of heterozygous null Haao, or Kynu, mice produced embryos in the expected Mendelian ratio of genotypes. Unexpectedly, all embryos were normal (Table 12, Table 13). The metabolites up and downstream of HAAO, or KYNU, in adult mouse serum were quantified. Haao-/- mice had 3HAA levels >100-fold higher than Haao+I-, or Haao+l+, mice (Table 14). Similarly, Kynu-/- mice had 3HK levels >70-fold higher than Kynu+I-, or Kynu+I+, mice. This was consistent with findings in humans ( Figure 1 , Table 11 , Table 12). By contrast, NAD levels were the same in all mice regardless of genotype (Table 15). This suggested that elevated levels of metabolites upstream of HAAO, or KYNU, in humans were not causing congenital malformation. Rather, it indicated that reduced NAD level in humans is the cause of congenital malformation.
  • mice were intercrossed. Embryos were harvested at embryonic day (E) 18.5 from 10 Haao litters and 3 Kynu litters. Ratios of genotypes were tested for goodness of fit to expected Mendelian segregation (1 :2: 1 ) by ⁇ 2 analysis, calculated with two degrees of freedom (p). *Muscular ventricular septal defect, nd, not determined.
  • mice of specified genotype Female and male Haao, or Kynu, mice of specified genotype were crossed. Embryos were harvested at embryonic day (E) 14.5 or 18.5. Niacin-free diet (NFD) and niacin (nicotinic acid) in drinking water was given on specified days during gestation, a, one -/- with right thoracic 2-3 vertebral fusion; one -/- with kidneys 30% smaller than heterozygous littermates.
  • NFD nuclear factor-free diet
  • niacin nicotinic acid
  • mice were maintained on complete rodent diet containing 90 mg/kg niacin and 3.7g/kg tryptophan. Blood was drawn at 2 pm - 4 pm and serum analyzed. Values are expressed as meant standard deviation. NAD(H) is the sum of NAD+ and NADH. p: One way ANOVA test (mutant vs WT, Dunnett multiple comparison test ). ns: not significant. See Figure 2.
  • NAD is produced by two pathways that require the dietary input of tryptophan and niacin (Figure 2).
  • the NAD de novo synthesis pathway catabolizes tryptophan via the kynurenine pathway and the NAD salvage pathway converts niacin and other precursors into NAD (independent of KYNU and HAAO).
  • the NAD status of mice is known to be at least four times higher than humans, possibly because they convert tryptophan to NAD more efficiently, and they consume more tryptophan/niacin per body weight due to a higher basal metabolic rate.
  • embryos receive niacin from the mother as well as generating their own.
  • mice In mice, reduced niacin status only occurs when both de novo synthesis of NAD is blocked and niacin is removed from the diet.
  • NAD niacin-free diet
  • null females were mated with
  • Haao+I-, or Kynu+I- mice were intercrossed.
  • Females were placed on a niacin free diet (NFD) for the duration of gestation.
  • Embryos were harvested at embryonic day (E) 18.5 from 9 Haao litters and 7 Kynu litters. Ratios of genotypes were tested for goodness of fit to expected Mendelian segregation (1 :2: 1 ) by ⁇ 2 analysis, calculated with two degrees of freedom (p). "Ventricular septal defect, nd, not determined.
  • Haao-I- females were mated with Haao+I- males.
  • Kynu-I- females were mated with Kynu+I- males.
  • Females were placed on a niacin-free diet (NFD) for indicated durations during gestation. Embryos were harvested at embryonic day (E) 14.5. All embryos were resorbed and were unable to be genotyped.
  • NFD niacin-free diet
  • mice were fed as before, and water was supplemented with 10 or 15 mg/L nicotinic acid. Since mice consume 1.3 litres of water/kg of food we calculated that with this regimen mice would consume 14% of the niacin equivalent in complete mouse chow (90 mg/kg). Litters from these mice contained embryos with genotypes in the expected Mendelian ratio, with 10 mg/L nicotinic acid null embryos were normal except for kidneys that were 30% smaller than heterozygous controls, with 15 mg/L nicotinic acid all embryos were normal (Table 4). Consistent with the phenotypic rescue, the inventors also observed a dose dependent increase in embryonic NAD(H) in response to maternal niacin
  • Example 11 Embryo loss, birth defects, NAD deficiency, and vitamin B3 as a preventative
  • mice consumed 591.5 micrograms/day of NE;
  • mice are more efficient than humans at converting tryptophan to NAD;
  • a genetic block (KYNU/HAAO) in NAD synthesis from tryptophan that causes NAD deficiency and embryo loss and defects can be overridden with vitamin B3 supplementation.
  • NAD deficiency could occur with an adequate diet if the mother has a reduced ability to absorb or process nutrients required for NAD synthesis. For example, if she is affected by factors that alter the efficiency of NAD synthesis such as gastrointestinal infection or inflammation, depression, type 2 diabetes, and high body mass index.
  • Example 13 Dietary restriction causes NAD deficiency and adverse pregnancy outcomes.
  • NAD levels are 2.2 times lower in mouse embryos with defects than in controls, at E9.5.
  • wildtype live embryos that have defects only due to limited diet had NAD levels 1.5 times lower than controls, at E11.5 (data not shown).
  • Example 14 Genetic predisposition and dietary restriction cause adverse pregnancy outcomes.
  • Example 15 A genetic complementation assay in yeast identifies human NAD gene variants with impaired function.
  • 3-Hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • LC-MS/MS liquid chromatography-tandem mass spectrometry
  • Serum 3HK and 3HAA were detected and quantified using an Agilent 1290 UHPL37C system connected to an Agilent 6490 triple-quadrupole mass spectrometer. Briefly, 38neutralized samples (1 ⁇ L) were injected and separated on a 5 ⁇ m Luna C18 (2) column (3930 ⁇ 2.10 mm; phenomenex, USA) by gradient elution using mobile phase A (0.1 % ac40etic acid in H 2 0) and mobile phase B (0.1 % acetic acid in acetonitrile) at 0.15 mL min -1 .41 The gradient consisted of 0-5 % mobile phase B from 0 to 6 min, 5-100% from 6 to 8 42min.
  • VACTERL association The NAD de novo synthesis pathway catabolizes tryptophan leading to the production of NAD. Although metabolites upstream of the block are elevated and have postnatal functions, it is the deficiency in embryonic NAD,
  • NAD deficiency is linked to disease via both genetic and environmental means. Severe niacin deficiency causes pellagra characterized by dermatitis, diarrhea, dementia, and death. Although pellagra is rare today, niacin deficiency caused by dietary inadequacies, malabsorption of nutrients and drug interference is still observed. Niacin deficiency can also be inherited; Hartnup disease characterized by dermatitis, neurologic, and behavioral defects is caused by mutation of SLC6A19, required for transport of neutral amino acids including tryptophan. NAD synthesis is also reduced by pathophysiological factors such as type 2 diabetes, obesity, inflammation. These may have been confounding factors in some of the families studied. Niacin deficiency is also common during pregnancy; at least a third of women are deficient in niacin in the first trimester and double this by the third trimester.
  • genes encoding enzymes of the NAD salvage pathway (NAMPT, NMNAT1-3), those required for nicotinic acid transport (SLC5A8, SLC22A13), uptake of dietary niacin (nicotinic acid, nicotimamide) or nicotimamide riboside, or for their entry into the NAD producing pathways (NAPRT, PNP, NMRK1 ,2) could also be candidates for causing congenital malformations.
  • NAPRT, PNP, NMRK1 ,2 NAD producing pathways
  • mutation of NMNAT1 causes the retinal degenerative condition Leber congenital amaurosis type 9 (OMIM 608553) but not congenital malformation.
  • the largely normal enzymatic activity of mutant proteins would preclude a broad phenotype such as congenital malformation.
  • NAD is a co-factor with broad cellular utility involved in ATP production, PARP-dependent-DNA repair and sirtuin-dependent protein deacetylation.
  • NAD+/NADH participates in -400 redox reactions, NADP+/NADPH in -30 redox reactions, and NAD+ is degraded in -50 reactions involving PARP-dependent DNA repair or sirtuin-dependent deacetylation. Given this, there are many possible ways by which an NAD deficit might disrupt embryogenesis.
  • NAD is central to energy metabolism serving as an essential coenzyme in glycolysis, the tricarboxylic acid cycle, and the mitochondrial electron transport chain.
  • Embryogenesis requires metabolic "fluidity" between glycolysis and oxidative phosphorylation, as stem/progenitor cells transition from proliferation to differentiation. During embryogenesis, these transitions occur in spatially and temporally defined regions, with disruption likely to alter morphogenesis.
  • depletion of cellular NAD causes arrest of glycolysis, failure of ATP production, and loss of mitochondrial membrane integrity, resulting in cell apoptosis and necrosis.
  • sirtuin activity is required during embryogenesis for stem/progenitor cell fate decisions, and loss of activity causes multiple organ defects. Therefore an NAD deficit during embryogenesis could lead to defects due to reduced sirtuin activity.
  • DNA repair is active during rodent embryogenesis and is required for normal development. DNA base excision repair occurs via poly ADP ribosyl polymerase (PARP) activity, which is responsible for the majority of NAD catabolism.
  • PARP poly ADP ribosyl polymerase
  • Fanconi anemia a human DNA fragmentation disorder
  • VACTERL a human DNA fragmentation disorder

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Abstract

The invention relates to a method of preventing or treating a congenital malformation in an unborn offspring of a subject, the method comprising administering to the subject a therapeutically effective amount of NAD or an intermediate of NAD synthesis.

Description

DETECTION AND TREATMENT OF CONGENITAL MALFORMATIONS
Technical Field
[001] The technology relates to methods of detecting defects in nicotinamide adenine dinucleotide (NAD) synthesis that cause congenital malformations. The technology also relates to methods of treating congenital malformations caused by defects in NAD synthesis.
Related Application
[002] This application is based on and claims priority to Australian provisional patent application number 2017901409 filed 18 April 2017, the content of which is incorporated by reference in its entirety.
Background
[003] Major congenital malformations occur in at least 2% of human births. There are relatively few identified causes. The origins of most malformations have been difficult to determine, and possibly involve genetic and environmental factors, or gene- environment interaction. Although malformations frequently occur in isolation, they also occur in combination. VACTERL association describes a non-random combination of congenital defects without a known etiology. Individuals are typically categorized as having VACTERL association if they have any three of the following defects: vertebral defects (V), anal atresia (A), cardiac malformations (C), tracheoesophageal fistula with esophageal atresia (TE), renal dysplasia (R) and limb anomalies (L), in the absence of a known genetic cause (such as Fanconi anemia).
[004] Mutations in at least 100 genes have been shown to be responsible for numerous types of isolated heart defects. Isolated vertebral segmentation defects are currently known to be due to mutations in 7 genes, all associated with Notch signal transduction and somite formation. However, the genetic causes of isolated cardiac or vertebral defects have little relevance in cases where these defects occur in combination.
[005] The kynurenine pathway or de novo NAD synthesis pathway is the metabolic pathway which is responsible for the production of nicotinamide adenine dinucleotide (NAD) from the essential amino acid tryptophan. Two enzymes involved in the
kynurenine pathway are 3-hydroxyanthranilate 3,4-dioxygenase (HAAO) and
kynureninase (KYNU). HAAO catalyzes the synthesis of quinolinic acid from 3- hydroxyanth rani lie acid. KYNU catalyzes the hydrolysis of 3-hydroxy-L-kynurenine to 3- hydroxyanth rani lie acid and L-alanine. Another pathway by which NAD is produced is the NAD salvage pathway. In this pathway NAD is produced from nicotinamide and nicotinamide riboside. NAD can also be produced by the Preiss-Handler pathway. In this pathway NAD is produced from nicotinic acid. Some of these compounds are found in the diet but all are produced within cells and by metabolism of cellular NAD.
[006] Despite the presence of the de novo pathway, the NAD salvage pathway is essential in humans because there is a high cellular demand for NAD. The rate-limiting enzyme in the NAD salvage pathway is nicotinamide phosphoribosyltransferase.
[007] The present inventors have identified loss-of-function mutations in two genes (HAAO and KYNU) of the de novo NAD synthesis pathway. Each mutation causes multiple congenital malformations including defects in the heart and vertebrae. The present inventors have also developed methods to prevent or ameliorate a symptom of the malformations.
Summary
[008] In a first aspect, there is provided a method of preventing or treating a
congenital malformation in an unborn offspring of a subject, the method comprising administering to the subject an effective amount of NAD or an intermediate of NAD synthesis, wherein the congenital malformation is associated with or caused by at least one of:
a mutation in a gene encoding an enzyme involved in NAD synthesis; a dietary deficiency of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid;
inadequate absorption of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid; and
inadequate synthesis of NAD.
[009] The subject may be the mother of an unborn offspring, the unborn offspring or the born offspring.
[010] The intermediate of NAD synthesis may be selected from the group consisting of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, 2- amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, 2-aminomuconate, 2- oxoadipate, glutaryl-CoA, N-methylnicotinamide (MNA), deamido-NADN-methyl-4- pyridone-3-carboxamide (4-Py), N-methyl-2-pyridone-5-carboxamide (2-Py),
nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin, vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
[011] In some embodiments the intermediate of NAD synthesis is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinic acid
mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin, a derivative thereof, a salt thereof, and any combination thereof. For example the intermediate may be at least one of vitamin B3, nicotinamide or niacin (nicotinic acid).
[012] The NAD or intermediate of NAD synthesis may administered at a dose of at least about 0.1 mg/kg, at least about 0.3 mg/kg, at least about 1 mg/kg, at least about 25mg/kg, at least about 50 mg/kg, at least about 100 mg/kg, at least about 150mg/kg, or at least about 200mg/kg body weight of the subject.
[013] The NAD or an intermediate of NAD synthesis may be administered orally or by injection, for example by subcutaneous, intramuscular or intra-venous injection.
[014] The NAD or an intermediate of NAD synthesis may be administered before conception, around the time of conception or during the term of a pregnancy, or after birth if the NAD deficiency persists.
[015] In a second aspect there is provided use of an intermediate of NAD synthesis for the prevention or treatment of a congenital malformation in a unborn or unborn offspring of a subject, wherein the congenital malformation is associated with or caused by a at least one of:
a mutation in a gene encoding an enzyme involved in NAD synthesis;
a dietary deficiency of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid;
inadequate absorption of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid; and
inadequate synthesis of NAD.
[016] The intermediate of NAD synthesis may be selected from the group consisting of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, 2- amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, 2-aminomuconate, 2- oxoadipate, glutaryl-CoA, N-methylnicotinamide (MNA), deamido-NADN-methyl-4- pyridone-3-carboxamide (4-Py), N-methyl-2-pyridone-5-carboxamide (2-Py),
nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin (nicotinic acid), vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
[017] The intermediate of NAD synthesis may be selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide
mononucleotide, nicotinamide adenine dinucleotide, niacin, a derivative thereof, a salt thereof, and any combination thereof. For example the intermediate may be at least one of nicotinamide or niacin.
[018] In a third aspect there is provided a supplement comprising NAD or an intermediate of NAD synthesis when used for the prevention or treatment of a congenital malformation in a born or unborn offspring of a subject wherein the congenital malformation is associated with or caused by at least one of:
a mutation in a gene encoding an enzyme involved in NAD synthesis;
a dietary deficiency of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid;
inadequate absorption of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid; and
inadequate synthesis of NAD.
[019] The intermediate of NAD synthesis may be selected from the group consisting of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, 2- amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, 2-aminomuconate, 2- oxoadipate, glutaryl-CoA, N-methylnicotinamide (MNA), deamido-NADN-methyl-4- pyridone-3-carboxamide (4-Py), N-methyl-2-pyridone-5-carboxamide (2-Py),
nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin (nicotinic acid), vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
[020] In some embodiments the intermediate of NAD synthesis is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinic acid
mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin, a derivative thereof, a salt thereof, and any combination thereof. For example the intermediate may be at least one of vitamin B3, nicotinamide or niacin (nicotinic acid). [021] In some embodiments a dose of the supplement comprises about 15mg, 18mg, 20mg, 25mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1 ,000mg, 2,000mg or about 3,000mg of NAD or an intermediate of NAD synthesis.
[022] In a fourth aspect there is provided a method of detecting potential for a congenital malformation in a subject, the method comprising:
detecting a mutation in a gene encoding an enzyme involved in NAD synthesis from a sample from the subject,
wherein the presence of the mutation in the gene is associated with the congenital malformation.
[023] The subject may be one or both parents, an unborn offspring or a born offspring.
[024] In some embodiments the presence of the mutation is indicative of a predisposition to a congenital malformation in the subject's unborrn offspring.
[025] The congenital malformation may be selected from the group consisting of a vertebral defect, sacral agenesis, spinal dysraphism, a cardiac defect, a hypoplastic kidney, a solitary kidney, a shortened long bone; a limb abnormality, submucous cleft palate, bifid uvulua; laryngeal web, vocal cord palsy, anterior anus, restrictive lung disease, microcephaly, and any combination thereof.
[026] The gene may be selected from the group consisting of those listed in Table 20, namely aminoadipate aminotransferase, aminocarboxymuconate semialdehyde decarboxylase, acid phosphatase 5, arylformamidase, an alkaline phosphatase, aldehyde oxidase 1 , ADP-ribosyltransferase 1 , ADP-ribosyltransferase 2, ADP- ribosyltransferase 3, ADP-ribosyltransferase 4, ADP-ribosyltransferase 5, bone marrow stromal cell antigen 1 , catalase, CD38, cytochrome B5 reductase 3, cytochrome p450 family 2 subfamily D member 6, cytochrome p450 family 8 subfamily B member 1 , ectonucleotide pyrophosphatase/phosphodiesterase 1 , ectonucleotide
pyrophosphatase/phosphodiesterase 2, flavin adenine dinucleotide synthetase 1 , glutamate-ammonia ligase, 3-hydroxyanthranilate 3,4-dioxygenase, indoleamine 2,3- dioxygenase 1 , indoleamine 2,3-dioxygenase 2, kynurenine 3-monooxygenase, kynurenine aminotransferase 1 , kynurenine aminotransferase 3, kynureninase, leukosialin, NAD kinase, NAD kinase 2, NAD synthetase 1 , nicotinamide
phosphoribosyltransferase, nicotinate phosphoribosyltransferase, NAD(P)HX dehydratase, NAD(P)HX epimerase, nicotinamide nucleotide adenylyltransferase 1 , nicotinamide nucleotide adenylyltransferase 2, nicotinamide nucleotide adenylyltransferase 3, nicotinamide riboside kinase 1 , nicotinamide riboside kinase 2, nicotinamide N-methyltransferase, nicotinamide nucleotide transhydrogenase, 5', 3'- nucleotidase - cytosolic, 5'-nucleotidase - cytosolic I A, 5'-nucleotidase, cytosolic IB, NT5C1 B-RDH14 readthrough, 5'-nucleotidase - cytosolic II, 5'-nucleotidase - cytosolic IIIA, 5'-nucleotidase - cytosolic NI B, 5'-nucleotidase ecto, 5',3'-nucleotidase - mitochondrial, nudix hydrolase 12, poly(ADP-ribose) polymerase 1 , poly(ADP-ribose) polymerase 2, poly(ADP-ribose) polymerase family member 3, poly(ADP-ribose) polymerase family member 4, poly(ADP-ribose) polymerase family member 6, poly(ADP- ribose) polymerase family member 8, poly(ADP-ribose) polymerase family member 9, poly(ADP-ribose) polymerase family member 10, poly(ADP-ribose) polymerase family member 14, poly(ADP-ribose) polymerase family member 16, pyridoxal (pyridoxine, vitamin B6) kinase, purine nucleoside phosphorylase, pyridoxamine 5'-phosphate oxidase, prostaglandin I2 synthase, prostaglandin-endoperoxide synthase 2, quinolinate phosphoribosyltransferase, riboflavin kinase, sirtuin 1 , sirtuin 2, sirtuin 3, sirtuin 4, sirtuin 5, sirtuin 6, sirtuin 7, solute carrier family 3 member 2, solute carrier family 5 member 8, solute carrier family 6 member 19, solute carrier family 7 member 5, solute carrier family 7 member 8, solute carrier family 16 member 10, solute carrier family 22 member 13, solute carrier family 36 member 4, solute carrier family 52 member 1 , solute carrier family 52 member 2, solute carrier family 52 member 3, tryptophan 2,3-dioxygenase, tankyrase, tankyrase 2, and xanthine dehydrogenase.
[027] In some embodiments the gene may be selected from the group consisting of a gene encoding a tryptophan transporter, TD02 (tryptophan 2,3-dioxygenase), I DO 1 ,2 (Indoleamine 2,3-dioxygenase), AFMID (arylformamidase), KYNB (Kynurenine formamidase), KMO (kynurenine 3-monooxygenase), KYNU (kynureninase), HAAO (3- hydroxyanth rani late 3,4-dioxygenase), ACSMD (aminocarboxymuconate semialdehyde decarboxylase), QPRT (quinolinate phosphoribosyltransferase), NAPRT, NADSYN1 , NMAT1 , 2, or 3 (nicotinamide nucleotide adenylyltransferase 1 , 2, or 3), NADSYN1 (NAD synthetase 1), nicotinamide phosphoribosyltransferase. NMRK1 ,2 (Nicotinamide
Riboside Kinase 1 or 2), NAMPT (nicotinamide Phosphoribosyltransferase), PNP (Purine Nucleoside Phosphorylase), PARP1-6 ( Poly(ADP-Ribose) Polymerase 1-6), ART 1-4, SIRT1-7 (Sirtuin 1-7, also known as NAD-dependent deacetylase sirtuin 1-7), CD38 (cyclic ADP ribose hydrolase), BST1 (ADP-ribosyl cyclase 2), TRPT-1 (tRNA 2- phosphotransferase 1), and any combination thereof. [028] For example the mutation may be in HAAO or KYNU. In one embodiment the HAAO mutation is c.483dupT or c.559G>A. In another embodiment the KYNU mutation is c.170-1G>T, c.1045_1051 delTTTAAGC, or c.468>A.
[029] The mutation is detected by nucleic acid sequencing, multiplex ligation dependent probe amplification, single strand conformational polymorphism, or restriction fragment length polymorphism.
[030] The method may further comprise administering to the subject or the subject's mother an effective amount a supplement comprising at least one intermediate of NAD synthesis according to the third aspect.
[031] In a fifth aspect there is provided a method of detecting potential for a congenital malformation in a subject, the method comprising detecting at least one of:
a. a dietary deficiency of one or more of tryptophan, nicotinamide,
nicotinamide riboside, vitamin B3 and nicotinic acid;
b. inadequate absorption of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid; and
c. inadequate synthesis of NAD,
wherein the presence of any one of a, b or c is associated with the congenital malformation.
[032] The subject may be the mother of an unborn offspring or the unborn offspring.
[033] The presence of any one of a, b or c is indicative of a predisposition to a congenital malformation in the mother's unborn offspring.
[034] The congenital malformation is selected from the group consisting of a vertebral defect, sacral agenesis, spinal dysraphism, a cardiac defect, a hypoplastic kidney, a solitary kidney, a shortened long bone; a limb abnormality, submucous cleft palate, bifid uvulua; laryngeal web, vocal cord palsy, anterior anus, restrictive lung disease, microcephaly, and any combination thereof.
[035] The method may further comprise administering to the subject or the subject's mother an effective amount a supplement comprising at least one intermediate of NAD synthesis according to third aspect.
Definitions
[036] The terms 'treat' and 'treating' as used herein refer to one or more of the following:
(i) preventing development of a malformation (i.e. prophylaxis);
(ii) inhibiting or arresting development of the malformation;
(iii) relieving a malformation; and (iv) ameliorating, alleviating, lessening, or removing one or more symptoms of a malformation.
[037] The terms 'malformation' and 'congenital malformation' are used
interchangeably and refer to one or more of the following:
(i) an irregular, anomalous, abnormal, or faulty formation, structure or anatomical feature of an embryo, fetus, infant child or adult;
(ii) a neurocognitive deficit; and
(iii) a consequence of a malformation including miscarriage or spontaneous abortion.
[038] As used herein the term 'vitamin B3' refers to a mixture or complex comprising nicotinic acid (niacin), nicotinamide and nicotinamide riboside.
[039] It is to be noted that as used herein the term 'niacin' also refers to nicotinic acid.
[040] As used herein an 'intermediate of NAD synthesis' refers to any compound that is required or can be used, in the synthesis of NAD, including dietary sources such as nicotinic acid, nicotinamide, nicotinamide riboside, vitamin B3, and tryptophan.
[041] Throughout this specification, unless the context requires otherwise, the word 'comprise', or variations such as 'comprises' or 'comprising', will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
[042] The term 'consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[043] As used in this specification and the appended claims, the singular forms 'a', 'an', and 'the' include plural references unless the context clearly dictates otherwise. Thus for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.
[044] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this specification.
[045] In order that the present invention may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.
Brief Description of the Drawings
[046] Figure 1 : Families with congenital malformations have HAAO or KYNU mutations and altered plasma metabolites of NAD synthesis. Panel A shows pedigrees of families with congenital malformations. Symbols indicate male (square), female (circle), first trimester death/miscarriage (triangle), affected individual (patient, filled), deceased (slash). All individuals in families A, C and D were evaluated by exome sequencing. All individuals in family B were evaluated by genome sequencing. In family D two distinct variants in the KYNU gene were identified. Mutated alleles depicted as "a", "b", "c" or "d", "e", reference allele (+).
[047] Figure 2: NAD synthesis. Nicotinamide adenine dinucleotide (NAD) is synthesized, and levels maintained by, three pathways: (i) the NAD de novo synthesis pathway of NAD from dietary tryptophan occurs by the kynurenine pathway; (ii) the Preiss-Handler pathway from dietary nicotinic acid; (iii) the NAD salvage pathway from nicotinamide derived from the consumption of NAD and from the diet. Tryptophan, dietary niacin (vitamin B3) supplied as nicotinic acid and nicotinamide, and nicotinamide riboside, represent dietary inputs to NAD synthesis. The conversion of tryptophan via the kynurenine pathway occurs as depicted. 2-Amino-3-carboxymuconate-6-semialdehyde spontaneously converts to quinolinic acid. Quinolinic acid is converted to nicotinic acid mononucleotide (NaMN) by quinolinate phosphoribosyl transferase (QPRT). NaMN is converted to nicotinic acid adenine dinucleotide (NaAD) by nicotinamide nucleotide adenyltransferases (NMNAT1-3). NaAD is converted to nicotinamide adenine
dinucleotide (NAD) by NAD synthase 1 (NADSYN1). NAD is consumed and converted to nicotinamide (NAm), as it is a cofactor of ADP-ribosyltransferase reactions including poly(ADP-ribose) polymerase (PARP1-6), mono(ADP-ribosyl)- transferases (ART1-4), NAD+-dependent deacetylases (sirtuins; SIRT1-7), tRNA 2'-phosphotransferase
(TRPT1), and the second messenger producing ADP-ribosyl cyclases (CD38, BST1). As part of the NAD+ salvage pathway, NAm is converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT). NMN is converted to NAD by nicotinamide nucleotide adenylyltransferases (NMNAT1-3). Dietary nicotinic acid is converted to nicotinic acid mononucleotide (NaMN) by nicotinate
phosphoribosyltransferase (NAPRT). Dietary nicotinamide riboside is converted to nicotinamide (NAm) by purine nucleoside phosphorylase (PNP) or to nicotinamide mononucleotide (NMN) by nicotinamide riboside kinases (NMRK1.2). Nicotinamide (NAm) is methylated by nicotinamide-N-methyltransferase (NNMT) to N- methylnicotinamide (1-MNA). 1-MNA is oxidized and excreted in urine (not depicted). In some instances where the gene and protein names differ, the gene name and symbol are used.
[048] Figure 3. Low levels of NAD in null mouse embryos were rescued by niacin supplement. Haao, or Kynu, homozygous null female mice were mated with heterozygous male mice. Pregnant mice were on a niacin-free diet supplemented with 5, 10 or 15 mg/L nicotinic acid in drinking water from E7.5 to 9.5. Embryos were harvested at E9.5 and total NAD(H) levels quantified. The difference between groups was tested using One way ANOVA test (all other groups vs null on 5 mg/L nicotinic acid, Dunnett multiple comparison test, based on log2 transformed data). * p<0.05, *** p<0.001 , **** p<0.0001.
[049] Figure 4: Verification of HAAO and KYNU mutations in affected families.
Sanger DNA sequencing chromatograms show that HAAO c.483dupT (Family A; 11 -3) , HAAO c.558G>A (Family B; II-4), and KYNU c.170-1G>T (Family C; II-4) are
homozygous in the affected proband of each family. Unaffected family members are homozygous reference or heterozygous for the mutation. KYNU c.468T>A and KYNU c.1045_1051 delTTTAAGC are heterozygous in the affected proband (Family D; 11-1). The father (1-1) is heterozygous for KYNU c.468T>A. The mother is heterozygous for KYNU c.1045_1051 delTTTAAGC. See Figure 1 for pedigrees. The variable nucleotide(s) is specified (*).
[050] Figure 5: Genomic organization of HAAO and KYNU and mutation locations. Genomic organization of HAAO (upper) and KYNU (lower) and the DNA and protein variants identified in the patients. HAAO variants for patient A (black) and B (red). KYNU variants for patient C (black) and D (red).
[051] Figure 6: Mutant HAAO or KYNU proteins lack activity. A) Human his- FLAG-dual tagged HAAO wildtype (WT) and mutant proteins p.D162* (M1) and p.W186* (M2) were expressed in E. coli. The crude cell lysates were subjected to SDS-PAGE, and the resolved flag tagged proteins detected by Western blotting and quantified using purified human wildtype HAAO protein as a standard. (B) HAAO overexpressing bacterial lysates were assayed for 3-hydroxyanthranilate 3,4-dioxygenase activity and the reaction rates determined. (C) Specific activity of wildtype and mutant HAAO. (D) Human his- FLAG-dual-tagged KYNU wildtype (WT) and mutant proteins p.V57Efs*21 (M1), p.F349Kfs*4 (M2), p.Y156* (M3), p.T198A (M4) were expressed in E. coli. Recombinant proteins purified by immobilised metal ion affinity chromatography were resolved by SDS-PAGE, stained by Coomassie blue and the specific bands were quantified by using BSA standards. (E) Purified KYNU proteins were assayed for the kynureninase activity and the reaction rates determined. (F) Specific activity of wildtype and mutant KYNU. Enzyme activity for the same enzyme preparations was assayed three times on three different days. Specific band for the recombinant enzyme (*). Error bar: standard error of mean.
[052] Figure 7. Generation and genotyping of Haao and Kynu mice. Haao, and Kynu, null alleles were generated by CRISPR/Cas9 gene editing. (A) Strategy for generating a null allele of Haao. Exon 2 of Haao was deleted by two flanking CRISPR guide RNAs (red arrowheads) to cause a frameshift in exon 3 (red box). (B) Sequence and location of PAM (blue) and spacer (red) sequences relative to nucleotides of exon 2 (underlined). (C) Strategy for generating a null allele of Kynu. Exon 3 of Kynu was deleted by two flanking CRISPR guide RNAs (red arrowheads) to cause a frameshift in exon 4 (red box). (D) Sequence and location of PAM (blue) and spacer (red) sequences relative to nucleotides of exon 3 (underlined). Intronic DNA in italics. Exonic DNA underlined. Deleted sequence in small font.
[053] Figure 8: Analysis of HAAO and KYNU protein expression and activity in mutant mouse lines. (A,E) Western blot analysis of HAAO or KYNU protein from adult liver of Haao or Kynu wildtype (+/+), heterozygous null (+/-), and homozygous null (-/-) mice. (B, F) Quantification of HAAO, or KYNU, protein by western blot. (C,G) Enzyme reaction rate of HAAO or KYNU from adult liver of Haao or Kynu +/+, +/-, or -/- mice. (D, H) Specific activity of HAAO and KYNU enzymes. Three mice per genotype were analyzed except Kynu +/- for which two mice were analyzed. Error bar: standard deviation
[054] Figure 9: NAD deficiency during gestation causes multiple congenital malformations in mice. Haao -/-, or Kynu-/-, female mice were mated with
heterozygous male mice and exposed to niacin-limited diet from E7.5-E12.5. Embryos were examined at E18.5. Arrows in panel H, V: talipes; I, W: caudal agenesis; J, X: vertebral fusion; K, Y: ventricular septal defect; L, Z: small kidney; M, A': toe syndactyly; N, B': cleft palate. Scale bar: 1 mm.
[055] Figure 10: NAD-related genetic and environmental factors cause adverse pregnancy outcomes in a dose dependent manner. Miscarriage and congenital malformations are caused by recessively inherited gene mutations (dots 1-2), gene-environment interaction (dots 10-1 1), or diet alone (dots 7-8), Lines indicate that different thresholds of NE (niacin equivalent) cause miscarriage and congenital malformation with different maternal and embryo genotypes. Niacin equivalent is the amount of niacin derived from tryptophan and niacin. Mother genotype: homozygous null (dots 1 -4), heterozygous null (dots 9-12), wildtype (dots 5-8). Embryo genotype: W (wildtype); H (heterozygous null); N (homozygous null).
[056] Figure 11 : Miscarriage and congenital malformations can be caused by diet alone: A tryptophan-low and niacin-free diet causes embryo loss and defects in wildtype mice. Tryptophan is an essential for protein synthesis as well as NAD synthesis. To show that the defects were due to NAD deficiency and not generally due to tryptophan deficiency, the defects were prevented by adding niacin back to the diet. The incidence of viability and defects was different (p<0.0001) between diets (Fischer's exact test). Niacin equivalent (NE) is the amount of niacin derived from tryptophan and niacin. .
[057] Figure 12: Wildtype, but not mutant HAAO and KYNU rescue loss of Bna1 and Bna5 genes in yeast. A genetic complementation assay has been developed to test if variants/mutations in the human HAAO or KYNU genes function like wildtype human genes in mutant yeast. This assay can be used to test if variants in numerous human genes, that are required for NAD synthesis, alter protein function. Growth assay of Bna1 and Bna5 null yeast, transformed with wildtype (WT) and mutant HAAO and KYNU expression plasmids. Paired T-tests: **<0.01 , ***<0.0001.
Description
[058] The present inventors have identified mutations that disrupt NAD synthesis and cause congenital malformations. The discovery that such mutations cause congenital malformation is ground breaking and provides a basis for the use of niacin or other compounds, or intermediates of the NAD synthesis pathway to treat or prevent the malformations. In addition the invention also provides a basis for diagnostics to identify subjects that are at risk of developing the malformations.
[059] In particular, the technology relates to methods of detecting defects in the de novo NAD synthesis pathway and methods of treating congenital malformations caused by defects in the de novo NAD synthesis pathway. The present inventors have identified loss-of-function mutations in HAAO and KYNU genes of the de novo NAD synthesis pathway. Each mutation causes multiple congenital malformations including defects in the heart and vertebrae. The present inventors have also developed methods to treat the malformations. In various embodiments the treatment may include administration of compounds that are not formed due to the defects in the de novo NAD synthesis pathway. In some embodiments the compound may be niacin. Congenital malformations
[060] Various malformations are caused by or associated with defects in a NAD synthesis pathway such as the de novo NAD synthesis pathway or the NAD salvage pathway. The defects are caused or associated with mutations in one or more genes encoding enzymes of the pathway.
[061] In some embodiments the malformation is an irregular, anomalous, abnormal, or faulty formation, structure or anatomical feature of an embryo, fetus, infant child or adult.
[062] For example, defects in NAD synthesis may cause one or more
malformations selected from the group comprising:
• vertebral defects, in particular vertebral defects predominantly affecting the thoracolumbar spine;
• sacral agenesis;
• spinal dysraphism;
• cardiac defects, such as patent ductus arteriosus (PDA), atrial septal defect (ASD) and hypoplastic left heart (HLH);
• hypoplastic kidneys;
• solitary kidney;
• shortened long bones;
• limb abnormalities, such as talipes (club foot);
• sensorineural hearing loss;
• submucous cleft palate;
• bifid uvula;
• laryngeal web for example with persistent laryngeal tracheomalacia;
• vocal cord palsy, such as iatrogenic vocal cord palsy;
• anterior anus;
• restrictive lung disease, for example restrictive lung disease due to spondylocostal defects;
• postnatal growth defects;
• postnatal cognitive defects;
• microcephaly;
• extreme short stature;
• intellectual disability; and
• speech delay. [063] Some more specific examples of malformations caused by or associated with defects in NAD synthesis include the following:
• Hypoplastic dens and arch of vertebrae;
• Thoracic and lumbar segmentation defects;
• 11 pairs of ribs;
• 11 pairs of irregular ribs;
• Sacral agenesis, including sacral agenesis with spinal lipoma;
• Thoracic and lumbar segmentation defects for example butterfly vertebrae;
• Sacral tethered cord including sacral tethered cord with a terminal lipoma and/or spinal dysraphism;
• Fused ribs;
• Thoracic segmentation defects including cobblestone, hemivertebrae and lateral fusions;
• Sacral pit.
[064] In some embodiments the malformation may be a neurocognitive deficit. For example the neurocognitive deficit may be any deficit in a subject's perception, memory, association and recall in the thought process and behaviour. For example the
neurocognitive deficit may be a developmental delay, an intellectual disability, a delay in speech development or a delay in behavioural development.
[065] The malformations may occur in isolation or in any combination. In some embodiments the malformations occur in combination. In one or more embodiments the malformation may be a cardiac defect, vertebral defect, limb defect, or any combination of one or more thereof. In some embodiments the same defect in AD synthesis will cause different malformations or combination of malformations in different subjects.
[066] In some embodiments the term malformation is used to refer to the consequence of a physical or morphological malformation or neurocognitive deficit such as those set out above. For example one consequence of a malformation may be miscarriage for example a pregnant female may be gestating an embryo with a malformation.
Methods of detection
[067] Mutations in the genes encoding any one of the enzymes involved in NAD synthesis, for example via the de novo NAD synthesis pathway, Preiss-Handler pathway or the NAD salvage pathway may lead to defects in the pathway.
[068] The gene may be selected from the group consisting of any gene listed in Table 20, that is the gene may be selected from the group consisting of aminoadipate aminotransferase, aminocarboxymuconate semialdehyde decarboxylase, acid phosphatase 5, arylformamidase, an alkaline phosphatase, aldehyde oxidase 1 , ADP- ribosyltransferase 1 , ADP-ribosyltransferase 2, ADP-ribosyltransferase 3, ADP- ribosyltransferase 4, ADP-ribosyltransferase 5, bone marrow stromal cell antigen 1 , catalase, CD38, cytochrome B5 reductase 3, cytochrome p450 family 2 subfamily D member 6, cytochrome p450 family 8 subfamily B member 1 , ecto nucleotide
pyrophosphatase/phosphodiesterase 1 , ectonucleotide
pyrophosphatase/phosphodiesterase 2, flavin adenine dinucleotide synthetase 1 , glutamate-ammonia ligase, 3-hydroxyanthranilate 3,4-dioxygenase, indoleamine 2,3- dioxygenase 1 , indoleamine 2,3-dioxygenase 2, kynurenine 3-monooxygenase, kynurenine aminotransferase 1 , kynurenine aminotransferase 3, kynureninase, leukosialin, NAD kinase, NAD kinase 2, NAD synthetase 1 , nicotinamide
phosphoribosyltransferase, nicotinate phosphoribosyltransferase, NAD(P)HX
dehydratase, NAD(P)HX epimerase, nicotinamide nucleotide adenylyltransferase 1 , nicotinamide nucleotide adenylyltransferase 2, nicotinamide nucleotide
adenylyltransferase 3, nicotinamide riboside kinase 1 , nicotinamide riboside kinase 2, nicotinamide N-methyltransferase, nicotinamide nucleotide transhydrogenase, 5', 3'- nucleotidase - cytosolic, 5'-nucleotidase - cytosolic I A, 5'-nucleotidase, cytosolic IB, NT5C1 B-RDH14 readthrough, 5'-nucleotidase - cytosolic II, 5'-nucleotidase - cytosolic IIIA, 5'-nucleotidase - cytosolic NI B, 5'-nucleotidase ecto, 5',3'-nucleotidase - mitochondrial, nudix hydrolase 12, poly(ADP-ribose) polymerase 1 , poly(ADP-ribose) polymerase 2, poly(ADP-ribose) polymerase family member 3, poly(ADP-ribose) polymerase family member 4, poly(ADP-ribose) polymerase family member 6, poly(ADP- ribose) polymerase family member 8, poly(ADP-ribose) polymerase family member 9, poly(ADP-ribose) polymerase family member 10, poly(ADP-ribose) polymerase family member 14, poly(ADP-ribose) polymerase family member 16, pyridoxal (pyridoxine, vitamin B6) kinase, purine nucleoside phosphorylase, pyridoxamine 5'-phosphate oxidase, prostaglandin I2 synthase, prostaglandin-endoperoxide synthase 2, quinolinate phosphoribosyltransferase, riboflavin kinase, sirtuin 1 , sirtuin 2, sirtuin 3, sirtuin 4, sirtuin 5, sirtuin 6, sirtuin 7, solute carrier family 3 member 2, solute carrier family 5 member 8, solute carrier family 6 member 19, solute carrier family 7 member 5, solute carrier family 7 member 8, solute carrier family 16 member 10, solute carrier family 22 member 13, solute carrier family 36 member 4, solute carrier family 52 member 1 , solute carrier family 52 member 2, solute carrier family 52 member 3, tryptophan 2,3-dioxygenase, tankyrase, tankyrase 2, and xanthine dehydrogenase. [069] For example a gene encoding a tryptophan transporter, TD02 (tryptophan 2,3-dioxygenase), IDO 1 ,2 (Indoleamine 2,3-dioxygenase), AFMID (arylformamidase), KYNB (Kynurenine formamidase), KMO (kynurenine 3-monooxygenase), KYNU
(kynureninase), HAAO (3-hydroxyanthranilate 3,4-dioxygenase), ACSMD
(aminocarboxymuconate semialdehyde decarboxylase), QPRT (quinolinate
phosphoribosyltransferase), NAPRT, NADSYN1 , NMAT1 , 2, or 3 (nicotinamide nucleotide adenylyltransferase 1 , 2, or 3), NADSYN1 (NAD synthetase 1), nicotinamide phosphoribosyltransferase. NMRK1.2 (Nicotinamide Riboside Kinase 1 or 2), NAMPT (nicotinamide Phosphoribosyltransferase), PNP (Purine Nucleoside Phosphorylase), PARP1-6 ( Poly(ADP-Ribose) Polymerase 1-6), ART 1-4, SIRT1-7 (Sirtuin 1-7, also known as NAD-dependent deacetylase sirtuin 1-7), CD38 (cyclic ADP ribose hydrolase), BST1 (ADP-ribosyl cyclase 2), TRPT-1 (tRNA 2-phosphotransferase 1), and any combination thereof.
[070] In some embodiments the gene is HAAO or KYNU.
[071] The types of mutations that lead to defects in a NAD synthesis pathway include a point mutation such as a missense mutation, nonsense mutation or a synonymous mutation, a duplication, a deletion, an insertion or a splice mutation.
[072] The mutation may be homozygous or heterozygous.
[073] In embodiments where the mutation is heterozygous the malformation may not be as severe as the corresponding homozygous mutation. For example a subject with a heterozygous mutation may exhibit fewer malformations or a different combination of malformations than a subject with the corresponding heterozygous malformation. In some embodiments a subject with a heterozygous mutation may exhibit only a single malformation. Subjects with a heterozygous mutation may exhibit reduced severity of a malformation compared to a subject with a corresponding homozygous mutation.
[074] Defects in a NAD pathway can be detected by any means known in the art.
[075] For example, mutations can be detected using fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), PCR amplification of a gene or portion thereof and sequencing of the amplification product; sequencing such as whole-genome sequencing (WGS) or whole exome sequencing (WES), use of microarrays, multiplex ligation-dependent probe amplification (MLPA), single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis or restriction fragment length polymorphism (RFLP).
[076] Typically mutations are detected by exome or genome sequencing.
[077] Nucleic acid for mutation detection can be obtained from samples of blood, skin, amniotic fluid, chorionic villi. [078] In some embodiments defects in a NAD synthesis pathway can be detected by measuring the concentrations of metabolites produced by the enzymes of the pathway, either quantitatively or qualitatively. In another embodiment a defect a NAD synthesis pathway can be identified by measuring the level of NAD.
[079] In some embodiments the metabolites may be selected from the group comprising tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HAA), xanthurenic acid, quinolinic acid (also known as quinolinate), 2-aminomuconate, 2-oxoadipate, glutaryl-CoA, nicotinic acid mononucleotide, nicotinic acid adenine
dinucleotide (NaAD)(or deamido-NAD), nicotinamide adenine dinucleotide
(NAD), nicotinamide, N-methylnicotinamide (MNA), deamido-NADN-methyl-4-pyridone-3- carboxamide (4-Py), N-methyl-2-pyridone-5-carboxamide (2-Py), nicotinamide mononucleotide (NMN), a stable product derived therefrom (e.g. cinnabarinic acid), and any combination thereof.
[080] In embodiments where the sample is a urine sample the metabolite may be anthranilate, kynurenate, xanthurenate or any combination thereof.
[081] Any known methods can be used to measure NAD or a metabolite of NAD or a metabolite of NAD synthesis in a subject. For example NAD and metabolites can be detected, quantified or both using mass spectrometry, typically in conjunction with liquid chromatography. For example 3-hydroxykynurenine and 3-hydroxyanthranilic acid can be detected and quantified by liquid chromatography-tandem mass spectrometry (LC- MS/MS). Typically a serum sample is deproteinated with trichloroacetic acid before neutralisation. The sample is then applied to a C18 column and metabolites eluted using as gradient of 0-5 % mobile phase (e.g. acetic acid in acetonitrile). Selected fractions are then applied to a mass spectrometer for example a triple quadrupole mass spectrometer to detect 3HK and 3HAA by multiple reaction monitoring (MRM) in positive ion mode. The metabolite's can be quantified against commercial standards for example those available from Sigma Aldrich (USA).
[082] The measured concentration of NAD or a metabolite of the pathway can be compared to the concentration of NAD or a metabolite in a normal individual.
[083] NAD or metabolites can be measured in samples of blood, serum, plasma, saliva, urine, tear, lymph fluid, cerebrospinal fluid, mucosal secretion, peritoneal fluid, ascitic fluid, fecal matter, amniotic fluid, chorionic villus and body exudate.
[084] In some embodiments defects in NAD synthesis of a subject in utero can be detected by measuring the concentrations of metabolites in a maternal sample. For example, a sample of blood, serum, plasma, saliva, or urine, may be obtained from a pregnant female for analysis.
[085] Typically, NAD or metabolites of NAD synthesis or nicotinamide degradation are measured in a blood or urine sample.
[086] In some embodiments a defect in NAD synthesis is detected, for example by measuring metabolite levels or detecting a gene mutation, in a subject that is considering becoming pregnant. In this way the methods described herein can be used as prepregnancy testing for a defect in NAD synthesis.
Methods of Determining Risk of Malformation
[087] In some embodiments a subject may have a heterozygous mutation in a gene encoding an enzyme involved in NAD synthesis. In these embodiments, if that subject has sufficient dietary intake of, for example niacin, a malformation may not be apparent or the severity of a symptom associated with a malformation may be minimal. Without being bound by any particular theory it is believed that subjects with
heterozygous mutations may produce sufficient enzymes associated with NAD synthesis that sufficient NAD is produced at certain times, for example when the dietary intake of tryptophan, vitamin B6 (pyridoxal phosphate) or niacin is sufficient. However, if that subject is placed under stress such as for example dietary restriction of niacin or tryptophan, a digestive disorder that prevents the uptake of, for example vitamin B6, niacin or tryptophan such as often occurs during pregnancy, the subject may be at risk of for example increasing the severity of a symptom associated with a malformation. This is particularly the case for a subject's offspring when the offspring is in utero. For example a subject with a heterozygous mutation in a gene encoding an enzyme involved in NAD synthesis may normally produce sufficient NAD, however, in times of physiological stress such as restriction of vitamin B6, niacin or tryptophan that may occur during pregnancy, the offspring, who may also have a heterozygous mutation may be at greater risk of developing a malformation.
[088] A subject with a heterozygous mutation in a gene encoding an enzyme involved in NAD synthesis is at greater risk of producing an offspring with a malformation if both of the offspring's biological parents have a heterozygous mutation in a gene encoding an enzyme involved in NAD synthesis.
[089] Accordingly, the risk of a subject's offspring having a congenital malformation caused by a defect in NAD synthesis can be determined by assessing whether one or more of the offspring's biological parents have a defect in NAD synthesis. [090] For example a sample from one of the biological parents can be tested for a mutation in a gene encoding an enzyme involved in NAD synthesis. If a mutation is found, and is heterozygous then this is indicative of a predisposition to a congenital malformation in the subject's offspring.
[091 ] In another embodiment a sample from one of the biological parents can be tested the concentration of NAD, a metabolite of NAD synthesis or degradation, or a stable product from a metabolite in the sample. That concentration can be compared to the concentration of NAD, the metabolite or the stable product to a predetermined concentration of NAD, the metabolite or the stable product in a normal subject (i.e. one without a defect in NAD synthesis). If the concentration of NAD, the metabolite or the stable product is lower than that of a normal subject then that is indicative of a predisposition to a congenital malformation in the subject's offspring.
[092] In some embodiments the risk of a subject's offspring having a congenital malformation caused by a defect in NAD synthesis can be determined before conception of the offspring, for example by detecting defects in NAD synthesis using any of the methods disclosed herein.
[093] In embodiments where a subject is found to have, for example, a
heterozygous mutation in a gene encoding an enzyme involved in NAD synthesis or a metabolite or NAD concentration lower than a normal subject, treatment with a compound useful for the treatment of a congenital malformation caused by a defects in NAD synthesis is indicated to reduce the risk that the offspring will develop a
malformation or to reduce the severity of a symptom associated with a malformation in the subject whether that malformation is apparent or not.
Compounds
[094] In one or more embodiments, compounds useful for the treatment of congenital malformations caused by defects in NAD pathway (such as the de novo NAD synthesis pathway or the salvage pathway) are compounds, or salts or derivatives thereof, that are intermediates in the pathway downstream of the defect or the end product of the pathway, NAD or substrates and metabolites of the salvage pathway of NAD synthesis.
[095] Compounds useful for the treatment of a malformation include the following: tryptophan, N-formylkynurenine, kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, nicotinic
acid mononucleotide, nicotinic acid adenine dinucleotide (or deamido- NAD), nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin (nicotinic acid), vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
[096] Useful compounds for the detection of a defect in NAD synthesis include of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3- hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HAA), xanthurenic acid, quinolinic acid (also known as quinolinate), 2-aminomuconate, 2-oxoadipate, glutaryl-CoA, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide (NaAD)(or deamido- NAD), nicotinamide adenine dinucleotide (NAD), nicotinamide, N-methylnicotinamide (MNA), deamido-NADN-methyl-4-pyridone-3-carboxamide (4-Py), N-methyl-2-pyridone- 5-carboxamide (2-Py), and nicotinamide mononucleotide (NMN). Some of these compounds are known to form stable derivatives and so any stable product derived from these compounds can be used to detect defect in NAD synthesis. Any combination of the products can be used to detect defects in NAD synthesis.
[097] For example if the congenital malformation is caused by a defect in the KYNU gene then examples of compounds useful for the treatment of the malformation include 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconate-6-semialdehyde;
quinolinic acid, niacin, nicotinamide adenine dinucleotide, nicotinamide and nicotinamide mononucleotide.
[098] If the congenital malformation is caused by a defect in the HAAO gene, then examples of compounds useful for the treatment of the malformation include 2-amino-3- carboxymuconic semialdehyde; quinolinic acid, niacin, nicotinamide adenine
dinucleotide, nicotinamide and nicotinamide mononucleotide.
[099] In other embodiments the malformation may be treated by tryptophan, 3- hydroxyanth rani lie acid, 2-amino-3-carboxymuconic semialdehyde; quinolinic acid, niacin, nicotinamide adenine dinucleotide, nicotinamide, nicotinamide mononucleotide, derivatives thereof, salts thereof, and any combination thereof.
[0100] In one or more embodiments niacin is used to treat the malformation.
Compositions, Formulations and Supplements
[0101] A compound described herein can be prepared as a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of the disclosed compounds where the parent compound is modified by making an acid or base salt.
[0102] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. In other examples, conventional nontoxic salts include those derived from bases, such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like.
Pharmaceutically acceptable salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985).
[0103] The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
[0104] A compound described herein can be formulated in combination with one or more other compounds including another compound described herein.
[0105] A compound described herein can be formulated as a pharmaceutical composition or as a dietary supplement and administered to a mammalian host, such as a human patient or non-human animal, in a variety of forms adapted to the chosen route of administration.
[0106] A composition or supplement sometimes includes a diluent, buffer, preservative and the like. Various sustained release systems for drugs have also been devised, and can be applied to the compounds described herein.
[0107] Compounds described herein may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the subject's diet. For oral therapeutic administration, an active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, or wafers. Such compositions and preparations sometimes contain at least 0.1 % of active compound. The percentage of the compositions and preparations may be varied and sometimes are about 2% to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
[0108] Tablets, troches, pills, and capsules, may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
[0109] A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
[0110] A compound may be administered by infusion or injection (for example subcutaneous, intramuscular or intravenous injection). Solutions of a compound or a pharmaceutically acceptable salt thereof can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof. These preparations sometimes contain a preservative to prevent the growth of microorganisms.
[011 1] A dosage form can include a sterile aqueous solution or dispersion or sterile powder comprising an active ingredient, which are adapted for the preparation of sterile solutions or dispersions. The ultimate dosage form can be a sterile fluid which is stable under the conditions of manufacture and storage. A liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), nontoxic glyceryl esters, and suitable mixtures thereof. The prevention of the action of
microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. An isotonic agent, for example, a sugar, buffer or sodium chloride is included in some embodiments. Prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile solutions often are prepared by incorporating an active compound in a required amount in an appropriate solvent, sometimes with one or more of the other ingredients enumerated above, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods sometimes utilized are vacuum drying and the freeze drying techniques, which yield a powder of an active ingredient in addition to any additional desired ingredient present in the previously sterile-filtered solutions.
[0112] Dosage forms of some compounds such as niacin, nicotinic acid and nicotinamide are approved for therapeutic use in humans for treating niacin deficiency. However, the recommend daily allowance (RDA) for niacin is 16 milligrams per day for men and 14 milligrams per day for women. Conventional medical advice indicates that excess niacin or nicotinic acid can be harmful and may adversely affect liver function. In contrast the present inventors have found that doses of 50mg/kg of niacin for example, are useful in the treatment of malformations caused by defects in NAD synthesis.
[0113] In some embodiments the compound, such as niacin, may be administered as a controlled release tablet. For example a controlled release tablet may comprise about 5-30% high viscosity hydroxypropyl methyl cellulose, about 2-15% of a water- soluble pharmaceutical binder, about 2-20% of a hydrophobic component such as a waxy material, e.g., a fatty acid, and about 30-90% niacin.
[0114] A suitable controlled release tablet comprises: (a) about 5-20 percent by weight hydroxypropyl methylcellulose having a viscosity of about 10,000 CPS or greater, a substitution rate for the methoxyl group of about 7-30% and a substitution rate for the hydroxypropoxyl group of about 7-20%; (b) about 2-8 percent hydroxypropyl
methylcellulose having a viscosity of less than about 100, CPS methyl cellulose, or polyvinyl pyrollidone; (c) about 5-15 percent by weight hydrogenated vegetable oil or stearic acid; and (d) about 30-90% niacin.
[0115] The controlled released tablets can also be coated so as to further prolong the release of the niacin into the gastrointestinal tract, or to prevent its release into the stomach, in order to prevent or attenuate the gastrointestinal side effects which can accompany niacin administration.
[0116] For example, coatings predominantly comprising a polymeric material having a high degree of swelling on contact with water or other aqueous liquids can be used to further prolong the release of the compound from the tablet. Examples of suitable polymers include cross-linked sodium carboxymethylcellulose, cross-linked
hydroxypropylcellulose, hydroxymethylpropylcellulose, e.g., Methocel®,
carboxymethylamide, potassium methylacrylate divinylbenzene copolymer, polymethyl methacrylate, cross-linked polyvinylpyrrolidine, high molecular weight polyvinylalcohol, and the like.
[0117] Hydroxypropylmethyl cellulose is available in a variety of molecular weights and viscosity grades for example from Dow Chemical Co. under the Methocel® brand name. These polymers may be dissolved in suitable volatile solvents, along with dyes, lubricants, and flavorings, and coated onto the controlled release tablets, e.g., in amounts equal to 0.1-5% of the total tablet weight, by methods well known to the art. For example, see Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Publishing Co., Easton, Pa. (16th ed. 1980).
[0118] The controlled release tablets, for example niacin tablets, can be formulated to contain about 5mg, 10mg, 15mg, 20, mg, 30mg, 40mg, 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 825mg, 850mg, 875mg, 900mg, 925mg, 950mg, 975mg or about 1 ,000 mg, 1 ,250mg, 1 ,500mg, 1 ,750mg, 2,000mg, 2,250mg, 2,500mg, 2,750mg, 3,000 mg of the compound. The tablets are typically formulated for oral ingestion. In some embodiments they are sufficient to provide a total dosage of about 0.005 to 3.0g compound (e.g. niacin or vitamin B3).
[0119] In some embodiments the controlled release tablets will release about 10-35 wt-% of the total niacin within about 2 hours in an in vitro dissolution test, and about 40- 70 wt-% of the total niacin in eight hours.
[0120] In some embodiments, the concentration of a compound described herein in a liquid composition is about 0.1 wt %, 0.25wt %, 0.5wt %, 0.75wt %, 1wt %, 2wt %, 3wt %, 4wt %, 5wt %, 6wt %, 7wt %, 8wt %, 9wt %, 10wt %, 1 1wt %, 12wt %, 13wt %, 14wt %, 15wt %, 16wt %, 17wt %, 18wt %, 19wt %, 20wt %, 21wt %, 22wt %, 23wt %, 24wt %, 25wt %, 26wt %, 27wt %, 28wt %, 29wt %, 30wt %, 31wt %, 32wt %, 33wt %, 34wt %, 35wt %, 36wt %, 37wt %, 38wt %, 39wt %, 40wt %, 41wt %, 42wt %, 43wt %, 44wt %, 45wt %, 46wt %, 47wt %, 48wt %,49wt %, or 50wt %.
[0121] Useful dosages, whether by controlled release or not, of compounds can be determined by their in vivo activity in animal models, such as the mouse models described herein. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art. Treatment of Congenital Malformations
[0122] The compositions and formulation disclosed herein are useful for the treatment or prevention of malformations in a subject associated with or caused by defects in NAD synthesis. For example the defects may be due to mutations in one or more genes encoding enzymes active in NAD synthesis such as the genes listed in Table 20 which include genes encoding a tryptophan transporter, TD02 (tryptophan 2,3- dioxygenase), IDO 1 ,2 (Indoleamine 2,3-dioxygenase), AFMID (arylformamidase), KYNB (Kynurenine formamidase), KMO (kynurenine 3-monooxygenase), KYNU
(kynureninase), HAAO (3-hydroxyanthranilate 3,4-dioxygenase), ACSMD
(aminocarboxymuconate semialdehyde decarboxylase), QPRT (quinolinate
phosphoribosyltransferase), NAPRT, NADSYN1 , NMAT1 , 2, or 3 (nicotinamide nucleotide adenylyltransferase 1 , 2, or 3), NADSYN1 (NAD synthetase 1), nicotinamide phosphoribosyltransferase. NMRK1 ,2 (Nicotinamide Riboside Kinase 1 or 2), NAMPT (nicotinamide Phosphoribosyltransferase), PNP (Purine Nucleoside Phosphorylase), PARP1-6 ( Poly(ADP-Ribose) Polymerase 1-6), ART 1-4, SIRT1-7 (Sirtuin 1-7, also known as NAD-dependent deacetylase sirtuin 1-7), CD38 (cyclic ADP ribose hydrolase), BST1 (ADP-ribosyl cyclase 2), TRPT-1 (tRNA 2-phosphotransferase 1), and any combination thereof.
[0123] The malformations, may be for example cardiac malformations, vertebral, renal dysplasia, limb anomalies, and tracheoesophageal fistula, for example
tracheoesophageal fistula with esophageal atresia (TE).
[0124] Typically the malformations occur in the absence of a known genetic cause.
[0125] The term "therapeutically effective amount" as used herein refers to an amount of a compound provided herein, or an amount of a combination of compounds provided herein, to treat or prevent a malformation, or to treat a symptom of the malformation, in a subject.
[0126] As used herein, the terms 'subject' and 'patient' are used interchangeably to refers to an individual who will receive or who has received treatment (e.g.,
administration of a compound described herein) according to a method described herein, or who has a malformation caused by a defect in NAD synthesis, or who is at risk of having a malformation caused by a defect in NAD synthesis.
[0127] A compound described herein may be in a therapeutically effective amount in a formulation or medicament, which is an amount that can lead to a physiological effect (e.g., inhibiting the malformation), or lead to ameliorating, alleviating, lessening, relieving, diminishing or removing symptoms of the malformation, for example. [0128] A compound described herein, can be administered to any subject having a malformation caused by a defect in NAD synthesis. Examples of a subject include mammal, human, ape, monkey, ungulate (e.g., equine, bovine, caprine, ovine, porcine, buffalo, camel), canine, feline, rodent (e.g. mouse, rat). A subject may be male or female. The compound can be administered to a subject in any age group, including, for example, neonate, infant, juvenile, paediatric, adolescent and adult. In some
embodiments, for example where the subject is in utero the compound can be
administered to the pregnant mother of the subject.
[0129] In some embodiments a compound described herein can be administered to any subject to reduce the risk of a malformation caused by a defect in NAD synthesis. In another embodiment a compound described herein, can be administered to any subject to increase the amount of NAD in the subject to reduce the risk of a malformation caused by a defect in a NAD synthesis pathway, or to prevent such a malformation becoming apparent. For example a compound described herein can be administered to a subject who is, or is contemplating becoming pregnant in order to reduce the risk of a
malformation. In some embodiments a compound described herein can be administered to a subject who is, or is contemplating becoming pregnant in order to reduce the risk of miscarriage, for example miscarriage caused by a malformation.
[0130] Typically the methods involve administering a therapeutically effective amount of a compound to the subject with the malformation. The compound may be administered by any route, For example the compound may be administered by oral, parenteral, intravenous, intramuscular, topical, subcutaneous or intradermal routes.
[0131] The route of administration will vary depending on the nature of the compound and the way it is formulated. A skilled person will be able to determine the appropriate route of administration.
[0132] In some embodiments the subject is in utero and administration of the compound to the subject can be achieved by administering the compound to the mother of the subject.
[0133] In some embodiments the compound is administered by injection, for example a subcutaneous, intramuscular or intravenous injections.
[0134] In other embodiments the compound is administered orally.
[0135] The amount of the compound, or a salt or derivative thereof, required for treatment varies not only with a particular salt selected but also with the route of administration, the nature of the malformation being treated and the age and condition of the subject and will be ultimately at the discretion of the physician or clinician. [0136] In general a suitable dose is in the range of from about 0.1 mg to about 200 mg/kg, e.g., from about 50 to about 125 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, and often is in the range of 6 to 90 mg/kg/day, or about 15 to 60 mg/kg/day. A suitable dose, in general, sometimes is in the range of from about 1 to 150 mg/kg body weight of the recipient per day, e.g. from about 10 to about 130 mg/kg, from about 40 to about 120 mg/kg, from about 50 to about 100 mg/kg, from about 60 to 90 mg/kg, from about 65 to 85 mg/kg, or, for example, about 80 mg/kg/day.
[0137] The compound may be administered at a dose of at least about 50mg/kg body weight of the subject. For example the compound may be administered at a dose of about 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45,46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 11 1 , 112, 113, 114, 115, 1 16, 1 17, 1 18, 1 19, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, or 200 mg/kg body weight of the subject.
[0138] A compound may be conveniently administered in unit dosage form.
[0139] A desired amount of compound may be administered in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four, five, six, seven, eight, nine, ten or more than ten doses per day.
[0140] In embodiments where the compound is niacin a suitable dose is significantly more than the RDA for that compound. For example the dose may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, or 170 or more times the RDA for that compound.
EXAMPLES
Example 1 : Study Participants
[0141] Patients with congenital vertebral and heart malformations from Families A-C were identified by clinical geneticists at the Children's Hospital at Westmead and
Liverpool Hospital (Australia). Family D underwent diagnostic sequencing at GeneDx (USA) and was subsequently identified via GeneMatcher (Figure 1). Patients A-C were part of a cohort of 13 (3 consanguineous) families with multiple congenital malformations including vertebral and heart defects. Individuals from all 13 families underwent sequencing.
Clinical Features
[0142] Family A: Patient A was the male twin of a dizygotic pregnancy to first cousins once removed from Iraq. The mother had a previous miscarriage at 12 weeks gestation, with possible spinal defects. The other four children in the family are healthy.
[0143] There was no history of maternal diabetes or known teratogen exposure during the pregnancy. The twins were born at 34 weeks gestation. Patient A had a birth weight of 2240 g (42nd percentile), 760 g lighter than his unaffected twin sister, and occipitofrontal circumference (OFC) 31 cm (30th percentile). Apgar scores were 4 and 6 at one and five minutes respectively. He had a normal male karyotype. The patient had vertebral segmentation defects throughout the thoracic and lumbar spine that were associated with 11 pairs of irregular ribs. Other vertebral anomalies include hypoplasia of the arch of cervical (C) 1 and the dens, and sacral agenesis. Patient A had a primary atrial septal defect that required surgical repair. He had a submucous cleft palate, a bifid uvula, and laryngeal web with laryngotracheomalacia. He had a hypoplastic right kidney with vesicoureteric reflux. There were bilateral talipes with shortened arthrogrypotic feet requiring surgery. He had bilateral sensorineural deafness with Mondini defect and absent lateral semicircular canal, and recurrent otitis media. He was microcephalic by age 3 years, with OFC 45.8cm (Z score -2.5). At 12 years of age, he had marked short stature with height 120cm (<1st percentile, Z score -4.40), weight 28.8 kg (Z score - 2.28). He had global developmental delay with moderate intellectual disability and behavioral difficulties.
[0144] Family B: Patient B was the female twin of a dizygotic pregnancy, born at 38 weeks gestation, to first cousins from Lebanon. Her male twin and two older brothers are well and there is no family history of congenital heart disease, congenital anomalies or developmental delay. Her mother had gestational diabetes requiring insulin in the second half of the pregnancy.
[0145] Patient B was antenatally diagnosed with hypoplastic left heart (HLH). She was born at term with a birth weight of 2680 g (13th percentile), 270 g lighter than her male twin, and OFC 31.2cm (1st percentile). Apgar scores were 9 and 9 at one and five minutes respectively. Chromosome microarray detected no malformations. Postnatal echocardiogram confirmed the HLH with mitral stenosis, aortic stenosis and a
hypoplastic aortic arch. In the first week of life she underwent the Norwood procedure with the Sano shunt modification. Spinal imaging showed multiple thoracolumbar segmentation defects with abnormal rib configuration and butterfly vertebrae and a hypoplastic distal sacrum with sacral spinal dysraphism, tethered cord and terminal lipoma. She had a small dysplastic right kidney with normal renal function. She had left- sided moderate sensorineural hearing loss, no imaging of the auditory system was performed. She had a weak cry and dysphagia due to left vocal cord palsy, likely due to intraoperative damage of the recurrent laryngeal nerve. No laryngeal structural anomalies were detected endoscopically. At age 12 weeks, she was assessed as high risk for future cerebral palsy and movement problems, and early intervention was recommended. At 1 1 months of age, she died from complications of HLH.
[0146] Family C: Patient C was one of 13 pregnancies to first cousins from
Lebanon, eight of which were lost in the first trimester. The other siblings are alive and well. The mother had insulin-requiring diabetes and had a pre-pregnancy BMI of 32.8 kg/m2.
[0147] Congenital malformations were detected in the antenatal ultrasound scans, including short long bones, unilateral foot talipes, and hemivertebrae of the thoracic spine. The female baby was born at 39 weeks gestation with a birth weight of 3715 g (83rd percentile) and OFC 32.5cm (3rd percentile). Apgar scores were 9 and 9 at one and five minutes respectively. Postnatal X-rays showed prominent cobblestone segmentation defects in the thoracic spine and lumbar (L) 1 , with hemivertebrae and lateral fusions. These vertebral defects were associated with 1 1 pairs of ribs. The baby had normal female genitalia with a short perineum and anterior anus. Echocardiogram demonstrated a patent ductus arteriosus (PDA) that worsened with time, and led to increased requirements for respiratory support; surgical intervention was not performed. Abdominal ultrasound revealed small kidneys. The baby had no radial ray defects but had mild rhizomelic shortening, right talipes equinovarus, and bilateral 2-3 toe syndactyly. She had low-set ears, a small anterior forehead, upslanting small palpebral fissures and a short neck. The baby died at 4 months of age of restrictive lung disease due to thoracic spondylocostal defects.
[0148] Family D: Patient D was the first live-born child to non-consanguineous North American parents. She was preceded by two first trimester losses. She has a healthy younger sister. There was no known teratogen exposure during the pregnancy, but the mother had a BMI of 29.3 kg/m2.
[0149] Congenital heart disease, narrowed chest, hemivertebrae, short long bones and frontal bossing were detected antenatally and confirmed postnatally. The female patient was born at 38 weeks gestation with weight 2460 g (12th percentile), and OFC 37 cm (67th percentile). She had HLH for which she had surgery. There were vertebral segmental defects affecting thoracic (T) 7 to L1 , but the ribs were normal. She had a solitary left kidney with mild parenchymal changes, and Grade 3 kidney disease. She had hypothyroidism in infancy. At 38 months, she had a gastrostomy tube and failure to thrive and short stature, with height 74.9 cm (<1st percentile, Z score -5.3), weight 9.3 kg (<1st percentile, Z score -4.3), head circumference 49 cm (56th centile, Z score 0.17). She also had speech delay but no cognitive delay.
[0150] Written informed consent was obtained for all patients and their families, and the study was approved by the ethics committee at each participating institution.
[0151] With reference to Figure 1 , Table 1 summarizes patient clinical features, the identified DNA and protein variants, and fold changes in the levels of upstream (3HAA, 3HK) and downstream (NAD) metabolites in patient plasma compared with unaffected heterozygous family members. ASD (atrial septal defect); HLH (hypoplastic left heart); PDA (patent ductus arteriosus); VUR (vesicoureteric reflux); SNHL (sensorineural hearing loss); GDD (global developmental delay); ID (intellectual disability); HAAO (3- hydroxyanth rani late 3,4-dioxygenase); KYNU (kynureninase); 3HAA (3- hydroxyanth rani lie acid); NAD (nicotinamide adenine dinucleotide); na (not available); "+" denotes present; "-" denotes absent. NAD(H) is the sum of NAD+ and NADH. See Figures 2-5, Table 2, Table 12.
Figure imgf000032_0001
Figure imgf000033_0001
Example 2: Genomic And Genetic Analyses
[0152] All families underwent whole exome sequencing except for Family B where whole genome sequencing was employed. Methods of library preparation, sequencing, variant detection and re-sequencing are described below
DNA sequencing
[0153] Family A (individuals 1.1 , I.2, II.3, II.5) and Family C (individuals 1.1 , 1.2, II.4): DNA sequencing libraries were made using the Nimblegen SeqCap EZ Human Exome Kit v3.0 (Roche). Sequencing was performed using the lllumina HiSeq2000 to generate 100 bp paired-end reads (2 χ 100PE). Twelve samples were run per flow cell lane using version 3 SBS reagents (University of Queensland Diamantina Institute, Australia).
[0154] Family B (individuals 1.1 , 1.2, II.3, II.4): DNA sequencing libraries were made using the TruSeq Nano DNA HT Sample Prep Kit (lllumina Inc., California, CA, USA). Following clustering of each library on a single lane of a V2.5 patterned flowcell, paired- end sequencing with 150 bp read length was performed using the lllumina HiSeq X, (Garvan Institute, Australia).
[0155] Family D (individuals 1.1 , 1.2, 11.1): DNA sequencing libraries were made using the Agilent SureSelectXT Clinical Research Exome kit. Massively parallel sequencing was performed using the lllumina HiSeq2000 to generate 100 bp paired-end reads (GeneDx, USA). Bioinformatics
[0156] Following exome sequencing, DNA sequence reads were aligned to the human reference genome (hg19) using the Burrows-Wheeler Aligner (BWA) to create compressed Binary Alignment Maps (BAM) files. SNV calling was performed on the BAM files using Genome analysis toolkit software (GATK), with option of HaplotypeCaller. This generated a genomic variant calling (gVCF) file for each sample. VCF files from multiple samples were then processed together via a Joint Genotyping call with GATK to produce one multi-sample VCF file for all DNA variants. Variants in the VCF were annotated with various metrics using ANNOVAR. A count for the number of homozygous variants in the Exome Aggregation Consortium (ExAC) database was incorporated with a custom script. The resulting ANNOVAR-annotation file was split into individual families using a custom script.
WGS bioinformatics
[0157] Quality control of the WGS (Whole Genome Sequencing) sequencing data was performed using FastQC. DNA sequence reads were aligned to the human reference genome (hg38) using the Burrows-Wheeler Aligner (BWA) to create Sequence Alignment/Map (SAM) files. Samtools was used to transform these into compressed Binary Alignment Maps (BAM) files as well as for sorting and indexing. Read duplicates were marked using Picard. Variants including Single Nucleotide Variants (SNVs), Multiple Nucleotide Variants (MNVs) and insertion/deletions (indels) were called on the BAM files of the family jointly using PLATYPUS. The resulting Variant Call Format (VCF) file was annotated using ANNOVAR. Gender and relatedness within the family pedigree were confirmed using PLINK6 and KING. PLINK was also used to calculate summary statistics for each individual for quality control of the variants. These included the total number of variants, number of heterozygous genotypes, genotyping rate, number of singleton variants, and transition/transversion ratio.
Variant analysis
[0158] Each family's ANNOVAR-annotated variant file was viewed using the VarSifter application. Variants were retained when reads were present for that site in all family members. Potential disease-causing variants were identified following a series of filtering steps based on: the predicted mode of disease inheritance; the variant's effect on the gene (non-synonymous); minor allele frequency using the variant frequencies of multiple control databases (1000 Genomes, ExAC, ESP6500) and pathogenicity predictions of multiple bioinformatics algorithms (i.e. Polyphen-2,8 SIFT, MutationTaster. Manual verification of variant calls was performed by visual inspection of the sequence in question using the Integrative Genomics Viewer (IGV).
Sanger sequencing
[0159] Sequencing of both DNA strands was performed with BigDye v3.1 cycle sequencing reagents (Applied Biosystems), and capillary electrophoresis was performed on a 3730 DNA Analyzer (Applied Biosystems) at the Ramaciotti Centre for Gene Function Analysis (Australia). Primer sequences are shown in Table 2.
Table 2: primer sequences
Figure imgf000035_0001
Example 3: Activity of Recombinant Human HAAO And KYNU
[0160] 3-Hydroxyanthranilate 3,4-dioxygenase (HAAO) catalyzes the conversion of 3-hydroxyanthranilic acid (3HAA) to 2-amino-3-carboxymuconic 6-semialdehyde
(ACMS). Kynureninase (KYNU) catalyzes the conversion of 3-hydroxykynurenine (3HK) to 3HAA (Figure 2). The enzyme activity of mutant HAAO, or KYNU, was determined as described below.
Mutagenesis and cloning
[0161] The human HAAO cDNA clone was purchased from Mammalian Gene Collection (Accession No: BC029510). The human KYNU cDNA clone was purchased from Origene (RC214932). Using these plasmids as templates, site-directed
mutagenesis was carried out to introduce specific mutations to the HAAO and KYNU cDNA using the KAPA HiFi PCR Kit (Kapa Biosystems) and appropriate mutagenic oligonucleotides. The wildtype and mutated HAAO and KYNU open reading frame were then cloned into pT7 MAT-Tag® FLAG®-1 Expression Vector (Sigma-Aldrich) at the Hindi 11 and EcoRI sites. The expression constructs were sequenced to confirm that no secondary mutations arose from PCR amplification.
Protein expression and purification
[0162] The expression constructs were transformed into the E. coli BL21 (DE3) strain. Bacteria were grown in 0.5 L LB media containing 100 μg/mL ampicillin at 37°C until OD600 reached 0.6. Protein expression was induced by adding 0.5 mM IPTG and the cultures were incubated at 16 °C for a further 16h. Bacteria were pelleted, resuspended in lysis buffer (100 mM Tris-HCI pH 8, 0.3 M NaCI, 20 mM imidazole, 1x complete™, EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM PMSF, 0.05% Tween 20) and lysed by sonication on ice for 50 sec. Additional 0.5% NP-40 was included in the lysis buffer for the bacteria expressing the HAAO protein. The lysate was centrifuged at 21 ,000 rcf for 15 min at 4°C and the supernatant was passed through a 0.22 μm filter. The his-tagged KYNU recombinant proteins were purified by immobilized metal ion affinity chromatography using the AKTApurifier liquid chromatography system (GE Healthcare) and a 1 ml_ HisTrap HP column (GE Healthcare). Bound protein was eluted with elution buffer containing 0.3 M NaCI, 100 mM Tris-HCI pH 8, 0.05% Tween 20, and a step-wise increasing concentration of imidazole from 20 mM to 250 mM. Peak fractions were analyzed by SDS-PAGE to determine the fraction that contained the highest amount of recombinant protein. The concentration of the recombinant protein was estimated on the Coomassie stained SDS-PAGE gel by quantifying the intensity of the specific bands against known amounts of BSA as a standard. The concentrations of the HAAO proteins in the bacterial crude lysates were estimated by western blotting against FI_AG-tag (1 : 1000, F1804, Sigma Aldrich) using known amounts of recombinant HAAO protein as a standard.
Enzyme assays
[0163] 3-Hydroxyanthranilate 3,4-dioxygenase (HAAO) activity was measured spectrophotometrically according to Walsh et al (1991 ).1 1 Briefly, 7 μL of bacterial lysates were loaded into a 96-well plate in duplicate wells. The reaction was initiated at room temperature by adding 200 μL of reaction mixture containing 0.3 mM
(NH4)2Fe(S04)2, 60mM MES pH 6.5, and 0.1 mM 3-hydroxyanthranilic acid (3HAA). The reaction was monitored by the increase in absorbance at 360 nm every 5 sec for 1 min with a PHERAstar plate reader (BMG Labtech). The specific activity of enzyme was defined as the amount of 2-amino-3-carboxymuconic 6-semialdehyde (ACMS) produced per mg of enzyme per min, using the extinction coefficient of 47,500 M-1cm-1 for ACMS. Kynureninase (KYNU) activity was measured fluorometrically in a 96-well plate as described by (Gaertner et a/ J Bacteriol 1971 ; 108(2): 902-9). Briefly, 50 μL of purified recombinant KYNU was added into a 96-well plate in duplicate wells. The reaction was initiated at room temperature by adding 200 μL of reaction mixture containing 4 μΜ pyridoxal phosphate, 0.1 mM 3-hydroxy-DL-kynurenine (3HK) in 10 mM Tris-HCI pH 8. The reaction was monitored fluorometrically every 5 sec for 1 min with a FLUOstar plate reader (BMG Labtech) using the excitation and emission wavelengths of 340 nm and 405 nm, respectively. The specific activity of enzyme was defined as the amount of 3- hydroxyanth rani lie acid (3HAA) produced per mg of enzyme per min, using known amounts of 3HAA as a standard.
Example 4: Quantification of Kynurenine Pathway Metabolites and NAD
[0164] Metabolites were quantified in human plasma using high-performance liquid chromatography and gas chromatography-mass spectrometry as described in the Supplementary Appendix. Metabolites were quantified in mouse serum using liquid chromatography-tandem mass spectrometry as described in the Supplementary
Appendix. Serum and tissue NAD were measured using the NAD/NADH-Glo™ Assay kit (Promega).
Example 5: Generation and Analysis of HA AO and KYNU Mutant Mice
[0165] Null mutations in Haao, or Kynu, were generated in mice using
CRISPR/Cas9 as described in the Supplementary Appendix. Mice were fed a complete diet (90 mg/kg niacin and 3.7 g/kg tryptophan) (Gordons Premium Breeder Rat & Mouse, Gordons Specialty Feeds, Bargo, Australia), or a niacin-free diet (NFD; 1.8 g/kg tryptophan) (SF16-049, Specialty Feeds, Glen Forrest, Australia) with 0, 5, 10 or 15 mg/L nicotinic acid in drinking water. Embryos were harvested at various stages and analyzed as described below.
Generation of null alleles in mice
[0166] CRISPR guide RNAs were designed using CRISPR Design tool
(crispr.mit.edu) and generated "in house" by cloning 20 nucleotide oligos into pX330-U6- Chimeric_BB-CBh-hSpCas9, which was a gift from Feng Zhang (Addgene plasmid # 42230) as described.14(Cong et al. Cell, 2013Cas9 mRNA was generated by IVT (mMessage) from pCMV/T7-hCas9 (Toolgen) digested with Xhol. CRISPR guide RNA (50ng^L) and guide RNA pairs (100 ng/μί each) were injected into C57BL/6N zygotes, transferred to pseudo pregnant recipients and allowed to develop to term.
[0167] Haao: Guide RNAs were designed with spacers targeting either side of exon 2 (5'-TGGTGACACGTTAATGCGTG-3\ 5'-CACTTCCTAG ACG AGTCCTA-3') . Founder pups were screened for exon deletion by PCR amplification across the targeted region (F-5'-GCCCAAGATTGCAATAAAGC-3\ R-5' -GTTCCTTTCCGTCCTCCATT-3') .
[0168] Kynu: Guide RNAs were designed with spacers targeting either side of exon 3 (5'-GGCATAACTTAAACCCACAG-3\ 5'-AAGTTACACCCTAGCATATT-3') . Founder pups were screened for exon deletion by PCR amplification across the targeted region (F-5'-GAAAGAGCAAAAGATCTAGAGACCA-3\ R-5' -CATCAGTGTCACCCAGCCTA- 3').
[0169] To generate Haao, or Kynu, mutant mice we used CRISPR/Cas9 genome editing to induce double stranded breaks flanking exon 2 of Haao, or exon 3 of Kynu- 001, with a view to create a frameshift mutation early in the open reading frame to disrupt protein function. Eleven founder mice were generated for Haao that deleted exon 2 as judged by PCR across the target exon. Five of these lines had a frameshift mutation confirmed by Sanger sequencing. Haao founder 4 was chosen for further analysis; it carries an allele with a 325 base pair deletion (c.82-167_161-4232del/p.His28Cysfs*8) that encodes for a truncated protein consisting of 28 N-terminal amino acids of HAAO. Eight founder mice were generated for Kynu that deleted exon 3 as judged by PCR across the target exon. Four of these lines had a frameshift mutation demonstrated by Sanger sequencing. Kynu founder 20 was chosen for further analysis; it carries an allele with a 409 base pair deletion (c.253-200_294-7827del/p.lso58Argfs*7) that encodes for a truncated protein consisting of 58 N-terminal amino acids of KYNU.
Genotyping mice
[0170] Mice and embryos were genotyped by PCR. DNA samples were prepared from tails, yolk sacs, or whole embryos as described previously (Sasaki et al. Journal of Biological Chemistry 2015;290(28): 17228-38). All mouse analyses were conducted by individuals blinded to their genotype.
Table 3: Primer Se uences
Figure imgf000038_0001
Figure imgf000039_0001
Verification of the knockout mice
[0171] Heterozygous null mice were intercrossed and liver harvested from the 6-8 weeks old offspring. The livers were lysed in 1x LDS sample buffer (Thermo Fisher Scientific) and subject to western blot analysis using the primary antibodies anti-Haao (1 : 1500, NBP1-77361 , Novus Biologicals), anti-Kynu (1 :500, 11796-1-AP, Proteintech) and anti-tubulin (1 :500, E3, Developmental Studies Hybridoma Bank) and the secondary antibodies Goat anti-rabbit AlexaFluor® 680 (1 :5000, Life Technologies) and Goat anti- mouse IRDye® 800 (1 :5000, LI-COR). Signals were detected using the Li-Cor Odyssey Infrared Imaging System. Bands were quantified using Gelanalyzer (gelanalyzer.com). The same liver tissues were lysed in PBS containing 1x complete Mini, EDTA-free Protease Inhibitor (Roche) at a ratio of 10 uL/mg liver tissue by sonicating on ice for 10 seconds. 5 μL of Haao liver and 10 μL of Kynu lysate were loaded for the enzyme activity assay as described previously. The enzyme activities were normalized against the wet weight of liver tissue.
Example 6: Clinical Features of Families with Congenital Malformations
[0172] The clinical features of the study participants are summarized in Figure 1 and Table 4. The four families include one consanguineous family from Iraq (Family A), two consanguineous families from Lebanon (Families B and C), and a family from North America (Family D) (Figure 1). There is no other history of congenital anomalies or intellectual disability in these families. The mother in Family B had insulin-requiring gestational diabetes. The mother in Family C had pre-pregnancy insulin-requiring diabetes, hypercholesterolemia, and a body mass index (BMI) in the obese range. The mother in Family D had a BMI borderline obese. Patients A and B were each a dizygotic twin.
Figure imgf000040_0001
[0173] All affected individuals were born with vertebral defects predominantly affecting the thoracolumbar spine. Also, Patients A and B both had a spinal lipoma, associated with sacral agenesis in Patient A and spinal dysraphism in Patient B. All patients had cardiac defects: patent ductus arteriosus (PDA) in Patient C; an atrial septal defect (ASD) in Patient A; and hypoplastic left heart (HLH) in Patients B and D. Patients A, B, and C had hypoplastic kidneys, and Patient D had a solitary left kidney with moderate chronic kidney disease. Patients C and D had shortened long bones. Patients A and C had talipes. Patients A and B had sensorineural hearing loss. Patient A had a submucous cleft palate, bifid uvula, and a laryngeal web with persistent laryngeal tracheomalacia. Patient B had left vocal cord palsy that was possibly iatrogenic. None of the patients had a tracheoesophageal defect. Patient C had an anterior anus. Patient C died at age 4 months of restrictive lung disease due to spondylocostal defects and Patient B at age 11 months due to HLH complications. In addition to congenital malformations, postnatal growth and cognitive defects were evident. Patients A to C were microcephalic. Patients A and D have extreme short stature. Patient A has moderate intellectual disability, and behavioral issues at age 12 years, and Patient D has speech delay at age 3 years.
Example 7: Identification of HA AO and KYNU Mutations in Patients with Multiple Congenital Defects
[0174] Genomic sequencing was used to identify disease-causing variants in coding exons and splice sites. In the consanguineous families variants were filtered according to a recessive inheritance model. For completeness, variants were also filtered according to a compound heterozygous inheritance model, or for a de novo mutation in the patient. Additional filtering selected variants that were non-synonymous, rare, and predicted to be damaging, and these variants were assessed for further evidence of disease causation (Tables 5-9). Variants that introduced a premature stop codon (stop-gain) or disrupted transcript splicing were considered more likely to be damaging than missense variants. Of these, variants in two genes (HAAO, KYNU) both associated with NAD synthesis (Figure 2) were identified in three consanguineous families and were prioritized for further analysis. Credible disease-causing variants in genes associated with NAD synthesis were not identified in the remaining 10 families. Neither HAAO or KYNU have been associated with congenital malformation; however, a KYNU missense mutation (p.T198A) is associated with hydroxykynureninuria (OMIM 236800).
Figure imgf000042_0001
Table 6. Family A Variant Analys
Figure imgf000043_0001
(1 ) de novo: minor allele frequency (MAF) < 0.01 (1 %) and < 1 homozygous alleles; recessive MAF < 0.01 (1 %) and < 5 homozygous alleles; compound heterozygous MAF < 0.1 (10%). MAF and the number of homozygous variants sourced from the Exome Aggregation Consortium (ExAC) database3 (Lek et al. , 2015). Human genome assembly used in this analysis was Genome Reference Consortium GRCh38 (hg38).
Figure imgf000044_0001
Mouse model, http://www.informatics.jax.org/ OMI M, online Mendelian inheritance in man,
https://www.ncbi. nlm.nih.gov/omim Human genome assembly used in this analysis was Genome Reference Consortium GRCh38 (hg38).
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
[0175] Sanger sequencing confirmed variant segregation with disease (Figure 1 , Figure 4). In Family A, the patient was homozygous for a c.483dupT variant in HAAO, leading to a stop codon (p.D162*). The parents and unaffected siblings were
heterozygous for this variant. In Family B, the patient was homozygous for a c.558G>A variant in HAAO leading to a stop codon (p.W186*). The parents and unaffected siblings were heterozygous for this variant. In Family C, the patient was homozygous for a c.170- 1G>T splicing variant in KYNU leading to a stop codon downstream (p.V57Efs*21). The parents were heterozygous for this variant and the unaffected siblings were either heterozygous or homozygous reference. In Family D, the patient was compound heterozygous for KYNU variants c.468T>A and c.1045_1051delTTTAAGC both of which result in stop codons (p.Y156* and p.F349Kfs*4). The father was heterozygous for KYNU c.468T>A, while the mother was heterozygous for KYNU c.1045_1051delTTTAAGC. The location of the mutations in the HAAO and KYNU genes and proteins are shown in Figure 5.
Example 8: Mutant HAAO and KYNU Proteins Lack Activity In Vitro
[0176] The identified stop-gain variants are predicted to lead to nonsense-mediated decay of the transcripts. In case this does not occur in vivo, we tested the activity of the truncated enzymes. For HAAO the conversion of 3HAA to ACMS was quantified and for KYNU the conversion of 3HK to 3HAA. All the identified HAAO and KYNU mutants, as well as KYNU p.T198A that is associated with hydroxykynureninuria but not congenital malformation were tested. The specific activity of both the truncated HAAO and KYNU enzymes was either completely lost or greatly reduced compared with wildtype (Figure 6, Table 10). By contrast, the activity of KYNU p.T198A, had 64% of wildtype enzyme activity.
Table 10. Mutant HAAO and KYNU proteins have greatly reduced enzyme activity
Figure imgf000048_0001
Figure imgf000049_0001
Example 9: Kynurenine Pathway Metabolites and NAD are Altered in Patients
[0177] It was predicted that loss of HAAO or KYNU activity would lead to increased patient plasma metabolite levels upstream of these enzymes and reduced levels downstream. Patients A and B (homozygous HAAO stop-gain) had upstream 3HAA levels 64- and 385-fold higher. In Patient A, downstream NAD+ levels were 3-fold lower than unaffected heterozygous family members and in Patient B downstream NAD(H) levels were 4-fold lower than unaffected heterozygous family members (Figure 1 , Table 11 , Table 12). Plasma was not available from Family C. Patient D (compound heterozygous for KYNU truncating mutations) had upstream 3HK levels 161 -fold higher and downstream NAD(H) levels 7-fold lower than unaffected family members.
Figure imgf000050_0001
Figure imgf000051_0001
Quantification of kynurenine pathway metabolites in human plasma
[0178] Prior to analysis, serum samples were deproteinized with trichloroacetic acid at a final concentration of 5% (w/v) in equal volume. Samples were incubated for 5min, vortexed and then centrifuge (4°C) for 10min at 12,000rpm. Supernatant was then extracted and filtered with syringe filters (0.22μm) ready for injection into analyzers. Concurrent quantification of TRP, KYN, 3HK, and 3HAA was carried out in accordance to method previously described. An Agilent 1290 infinity ultra-high performance liquid chromatography system coupled with temperature controlled autosampler and column compartment, diode array detector and fluorescence detector was used for the analysis of these metabolites with a 20μL sample injection volume.
[0179] Separation of metabolites was performed under stable temperature of 38°C for 12min, using 0.1 mM sodium acetate (pH 4.65) as mobile phase, with an isocratic flow rate of 0.75ml/min in an Agilent Eclipse Plus C18 reverse-phase column (2.1 mm x 150mm, 1.8μm particle size). 3HK and KYN were detected using UV wavelength at 365nM. TRP and 3HAA were detected using fluorescence intensity set at Ex/Em wavelength of 280/438 for TRP and 320/438 for 3HAA. Mixed standards of all metabolites were used for a six-point calibration curve in order to interpolate the quantity of the sample readout. Agilent Openl_AB CDS Chemstation (Edition C.01.04) was used to analyze the chromatogram.
[0180] The inter- and intra-assay coefficient of variation (CV) is within the acceptable range of 3-7%. PA and QA were concurrently detected using an Agilent 7890 gas chromatograph coupled with an Agilent 5975 mass spectrometer in accordance to method previously described.16. In brief, 50 μL of extracted samples containing deuterated internal standards were derivatized with trifluoroacetic anhydride and hexafluoroisopropanol for an hour at 60°C.
[0181] The derivatized compound is then dissolved in toluene with 1 μL of this solution injected onto a DB-5MS capillary column (Agilent Technologies, Inc, Santa Clara, CA). Analysis was carried out with mass spectrometer operating in electron capture negative ionization mode and simultaneously monitored for selected ions (m/z 273 for PA, m/z 277 for 2H4-PA, m/z 467 for QA and m/z 470 for 2H3-QA).
Concentration of PA and QA are interpolated from the established six-point calibration curve based on abundance count ratio of the metabolites to their corresponding deuterated internal standards within each samples. The inter- and intra-assay CV met a 7-10% acceptability criterion. Example 10: Mice Null for HA AO, or KYNU, Model the Disease when NAD is Limiting
[0182] Mice lacking exon 2 of Haao, or exon 3 of Kynu, which each result in a premature stop codon in the subsequent exon (Figure 7) were generated. Enzyme assays confirmed that the edited Haao and Kynu, alleles were null (Figure 8).
Intercrosses of heterozygous null Haao, or Kynu, mice produced embryos in the expected Mendelian ratio of genotypes. Unexpectedly, all embryos were normal (Table 12, Table 13). The metabolites up and downstream of HAAO, or KYNU, in adult mouse serum were quantified. Haao-/- mice had 3HAA levels >100-fold higher than Haao+I-, or Haao+l+, mice (Table 14). Similarly, Kynu-/- mice had 3HK levels >70-fold higher than Kynu+I-, or Kynu+I+, mice. This was consistent with findings in humans (Figure 1 , Table 11 , Table 12). By contrast, NAD levels were the same in all mice regardless of genotype (Table 15). This suggested that elevated levels of metabolites upstream of HAAO, or KYNU, in humans were not causing congenital malformation. Rather, it indicated that reduced NAD level in humans is the cause of congenital malformation.
Table 13. Genotype distribution and phenotype of embryos resulting from
intercrosses of heterozygous null Haao, or Kynu, mice
Figure imgf000053_0001
Haao+I-, or Kynu+I-, mice were intercrossed. Embryos were harvested at embryonic day (E) 18.5 from 10 Haao litters and 3 Kynu litters. Ratios of genotypes were tested for goodness of fit to expected Mendelian segregation (1 :2: 1 ) by χ2 analysis, calculated with two degrees of freedom (p). *Muscular ventricular septal defect, nd, not determined.
Table 14. Summary: NAD deficiency causes embryo death and defects
Figure imgf000054_0001
Female and male Haao, or Kynu, mice of specified genotype were crossed. Embryos were harvested at embryonic day (E) 14.5 or 18.5. Niacin-free diet (NFD) and niacin (nicotinic acid) in drinking water was given on specified days during gestation, a, one -/- with right thoracic 2-3 vertebral fusion; one -/- with kidneys 30% smaller than heterozygous littermates.
Table 15. Kynurenine pathway metabolites, but not NAD, are altered in mutant adult mouse serum
Figure imgf000054_0002
Adult mice were maintained on complete rodent diet containing 90 mg/kg niacin and 3.7g/kg tryptophan. Blood was drawn at 2 pm - 4 pm and serum analyzed. Values are expressed as meant standard deviation. NAD(H) is the sum of NAD+ and NADH. p: One way ANOVA test (mutant vs WT, Dunnett multiple comparison test ). ns: not significant. See Figure 2.
[0183] NAD is produced by two pathways that require the dietary input of tryptophan and niacin (Figure 2). The NAD de novo synthesis pathway catabolizes tryptophan via the kynurenine pathway and the NAD salvage pathway converts niacin and other precursors into NAD (independent of KYNU and HAAO). The NAD status of mice is known to be at least four times higher than humans, possibly because they convert tryptophan to NAD more efficiently, and they consume more tryptophan/niacin per body weight due to a higher basal metabolic rate. During development, embryos receive niacin from the mother as well as generating their own. It is therefore possible that high maternal niacin may protect the null embryos from developing NAD deficiency in mice, and in order to reveal the requirement of de novo NAD synthesis for embryogenesis, the maternal NAD status has to be reduced. In mice, reduced niacin status only occurs when both de novo synthesis of NAD is blocked and niacin is removed from the diet. As a first attempt to mimic the reduced NAD levels of humans, pregnant heterozygous null mice were placed on a niacin-free diet (NFD). In litters from Haao+/-, or Kynu+/-, intercrosses, embryos were present with the expected Mendelian ratio of genotypes, and were normal
(Table 16, Table 14). This suggested that the heterozygous null females on a NFD produce sufficient NAD from dietary tryptophan to sustain normal embryonic
development. Therefore to preclude maternal NAD production from the de novo synthesis pathway in addition to the salvage pathways, null females were mated with
Haao+/-, or Kynu+/-, males and placed on a NFD. All embryos died, regardless of genotype (Table 16, Table 13). This was also true when the NFD was limited from E0.5 to E4.5-E6.5 (Table 16, Table 13). It was determined that a NFD supplemented with 5 mg/L nicotinic acid in drinking water between E7.5 and E12.5 better sustained embryogenesis in null mothers. Despite a large number of resorptions, live null embryos were present (Table 17, Table 13). All Haao+/-, or Kynu+/-, embryos were normal. By contrast all Haao-/-, or Kynu-/-, embryos had multiple defects including vertebral segmentation defects, conotruncal defects, small kidney, cleft palate, talipes, syndactyly, and caudal agenesis (Figure 9, Table 17, Table 18). NAD levels were 2-fold lower in null mouse embryos compared with unaffected heterozygous littermates at E9.5 (Figure 3).
This indicates that in both mice and humans, loss of embryonic NAD leads to embryo defects and death. Importantly, it is the embryonic NAD deficit that causes defects rather than the maternal deficit, as null mothers produce normal heterozygous embryos (Table
2, Figure 9). Table 16. Genotype distribution and phenotype of embryos resulting from intercrosses of heterozygous null Haao, or Kynu, mice in combination with a niacin- free diet
Figure imgf000056_0002
Haao+I-, or Kynu+I-, mice were intercrossed. Females were placed on a niacin free diet (NFD) for the duration of gestation. Embryos were harvested at embryonic day (E) 18.5 from 9 Haao litters and 7 Kynu litters. Ratios of genotypes were tested for goodness of fit to expected Mendelian segregation (1 :2: 1 ) by χ2 analysis, calculated with two degrees of freedom (p). "Ventricular septal defect, nd, not determined.
Table 17. Embryos resulting from females null for Haao, or Kynu, on a niacin-free diet
Figure imgf000056_0001
Haao-I- females were mated with Haao+I- males. Kynu-I- females were mated with Kynu+I- males. Females were placed on a niacin-free diet (NFD) for indicated durations during gestation. Embryos were harvested at embryonic day (E) 14.5. All embryos were resorbed and were unable to be genotyped.
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
[0184] To prove that lack of NAD was disrupting embryogenesis, pregnant null mice were fed as before, and water was supplemented with 10 or 15 mg/L nicotinic acid. Since mice consume 1.3 litres of water/kg of food we calculated that with this regimen mice would consume 14% of the niacin equivalent in complete mouse chow (90 mg/kg). Litters from these mice contained embryos with genotypes in the expected Mendelian ratio, with 10 mg/L nicotinic acid null embryos were normal except for kidneys that were 30% smaller than heterozygous controls, with 15 mg/L nicotinic acid all embryos were normal (Table 4). Consistent with the phenotypic rescue, the inventors also observed a dose dependent increase in embryonic NAD(H) in response to maternal niacin
supplementation (Figure 3). This demonstrates that embryo death and defects are specifically due to NAD deficit in mice. Importantly, it shows that in mice niacin supplementation prevents the disruption of embryogenesis, and thus such
supplementation prevents cases of in utero death and congenital malformation in humans.
Example 11 : Embryo loss, birth defects, NAD deficiency, and vitamin B3 as a preventative
[0185] Kynu and Haao null mice have elevated levels of upstream metabolites but, NAD levels were normal and null embryos lacked defects when mothers were fed on standard mouse chow. NAD is synthesised de novo from tryptophan, and more directly from vitamin B3 (niacin/nicotinic acid, nicotinamide, nicotinamide riboside; Fig. 2). The lack of defects occurred because:
a) the mouse chow was highly enriched with tryptophan (14.43 mg daily intake), and niacin (350 micrograms daily intake). Tryptophan and vitamin B3 intake is expressed as 'niacin equivalents' (NE), with 1 NE equal to 60 mg tryptophan or 1 mg vitamin B3. With this enriched diet mice consumed 591.5 micrograms/day of NE;
b) mice are more efficient than humans at converting tryptophan to NAD;
c) the Kynu or Haao heterozygous null mothers were able to convert this abundant tryptophan to NAD and nicotinamide.
[0186] The Kynu and Haao homozygous null embryos were able to convert nicotinamide to NAD via the salvage pathway (Figure 2) and so developed normally. When pregnant null mice were given a standard tryptophan but niacin-free diet (1 17 micrograms/day NE only from tryptophan), all embryos died regardless of genotype (Figure 10, dot no. 1), but with increased NE due to added niacin (148 micrograms/day) only homozygous null embryos developed defects and these were the same defects as those in human (Figure 10, dots no. 2). Null mouse embryos generated under these conditions had reduced NAD levels, at E9.5. Increasing the mothers' daily NE intake via niacin in water, normalised NAD levels and prevented embryo loss and birth defects in a dose-dependent manner (Figure 10, dots 3 and 4).
[0187] A genetic block (KYNU/HAAO) in NAD synthesis from tryptophan that causes NAD deficiency and embryo loss and defects can be overridden with vitamin B3 supplementation. This demonstrates that genetic and environmental factors combine to different extents, to cause, or prevent, adverse pregnancy outcomes. It indicates that NAD deficiency can be induced with other combinations of genetic and environmental factors. It also suggests that mutation of other genes that affect NAD levels might cause NAD deficiency and adverse pregnancy outcomes. The mice studies disclosed herein address the effects of differing contributions of environmental and genetic factors in causing NAD deficiency in gestation. For example, in C57BL/6 wildtype mice a dietary restriction alone caused NAD deficiency and characteristic embryo loss and defects, by restricting tryptophan and niacin (Figure 10, dots 7 and 8; Example 12, Figure 1 1). In a gene-environment model, a combination of a diet less deficient in tryptophan than that used with wildtype mice above and a genetic component, induced characteristics adverse pregnancy outcomes (Example 13 and Figure 10 - dots 10, 1 1 , 12). Importantly, NAD deficiency could occur with an adequate diet if the mother has a reduced ability to absorb or process nutrients required for NAD synthesis. For example, if she is affected by factors that alter the efficiency of NAD synthesis such as gastrointestinal infection or inflammation, depression, type 2 diabetes, and high body mass index.
Example 13: Dietary restriction causes NAD deficiency and adverse pregnancy outcomes.
[0188] In order to define dietary conditions that have the same effect as the Kynu or Haao genetic block, wildtype C57BL/6 female mice were placed on tryptophan-low diets (to reduce the amount of NAD synthesised from tryptophan) and a niacin-free diet (to ensure that NAD is synthesised only from tryptophan). Adverse pregnancy outcomes were observed in a dose dependent manner (Figure 10, dots 5-8). The defects were the same as those caused by the genetic block in NAD synthesis from tryptophan (Figure 9). A daily intake of 134.4 or 103.3 micrograms of NE did not cause adverse pregnancy outcomes (Figure 10, dots 5 and 6). By contrast, 36.2 micrograms of NE caused complete embryo loss (42/42) by E18.5 (Figure 10, dot 7). An increase to 41.3 micrograms of NE caused loss of 24/37 embryos, and defects in all 13 live embryos at late gestation (E18.5) (Figure 1 1 and Figure 10: dot 8). To ensure that embryo loss and defects were caused by NAD deficiency and not insufficient tryptophan for protein synthesis, the diet was supplemented with niacin (0.093mg/day) (NE 134.3 micrograms) and this prevented all embryo loss and almost all defects (only 2 of 23 embryos had defects) (Figure 11). Thus, we show in wildtype mice that a diet with 36.2-41.4 micrograms/day of NE cause adverse pregnancy outcomes typical of NAD deficiency. NAD levels are 2.2 times lower in mouse embryos with defects than in controls, at E9.5. In this example wildtype live embryos that have defects only due to limited diet, had NAD levels 1.5 times lower than controls, at E11.5 (data not shown).
Example 14: Genetic predisposition and dietary restriction cause adverse pregnancy outcomes.
[0189] Adverse pregnancy outcomes are induced with incomplete penetrance when genetic predisposition is combined with decreasing dietary NE in a dose dependent manner. After mating, Haao+I- (heterozygous null) female mice were placed on a tryptophan-low diet and a niacin-free diet. A daily intake of 1 17 microgram/day NE had no impact on embryogenesis with all embryos alive and normal, regardless of genotype (Figure 10: dot 9). By contrast, 62 microgram/day NE resulted in embryo loss and defects with complete penetrance in homozygous null embryos (Figure 10: dot 10), with reduced penetrance in heterozygous null embryos (Figure 10: dot 11), and a further reduction in penetrance in wildtype embryos (Figure 10: dot 12).
Example 15. A genetic complementation assay in yeast identifies human NAD gene variants with impaired function.
[0190] There are 95 human genes associated with NAD synthesis or metabolism, encoding for enzymes, and transporters of enzyme substrates and co-factors. A list of the 95 genes is shown in Table 20. The yeast knockout collection described in Winzeler et al. (Science. 1999;285(5429):901-6) contains null strains for -50 % of these human orthologues. Using these yeast strains an assay was developed to test the function of human gene variants. To prove the efficacy of this approach, using yeast lacking the HAAO or KYNU orthologues Bna 1 or Bna5 respectively, growth and intracellular NAD concentrations were quantified in the absence of niacin. Homozygous diploid yeast strains lacking Bna1 or Bna5 did not grow in medium lacking niacin and their intracellular NAD levels were 48 times lower than those of wildtype yeast. Introduction and expression of wildtype human HAAO or KYNU cDNA in Bna1 or Bna5 null yeast respectively, rescued the growth and NAD levels of these null strains, (Figure 12).
Consistent with the disclosure of this specification variants (HAAO M1 , M2 and KYNU M1 , M2, M3) that lacked enzyme activity and caused birth defects, were unable to rescue the yeast null strains (Figure 12).
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Quantification of kynurenine pathway metabolites in mouse serum
[0191] 3-Hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To measure the concentration of 3HK and 3HAA in mice serum, samples (50 μL) were de- proteinated by adding 12.5 μL of 20% trichloroacetic acid (w/v), left on ice for 15 min before centrifugation at 17,000 g, 4°C for 5 min. To 20 μL of supernatant, an equal volume of 1 M potassium phosphate pH 7.4 was added to neutralize the solution. Serum 3HK and 3HAA were detected and quantified using an Agilent 1290 UHPL37C system connected to an Agilent 6490 triple-quadrupole mass spectrometer. Briefly, 38neutralized samples (1 μL) were injected and separated on a 5 μm Luna C18 (2) column (3930 χ 2.10 mm; phenomenex, USA) by gradient elution using mobile phase A (0.1 % ac40etic acid in H20) and mobile phase B (0.1 % acetic acid in acetonitrile) at 0.15 mL min-1.41 The gradient consisted of 0-5 % mobile phase B from 0 to 6 min, 5-100% from 6 to 8 42min. Flow was then directed into the triple quadrupole mass spectrometer with pa4rameters set as follows: gas temperature = 250°C; gas flow = 20 L min-1; nebulizer pressure = 35 psi; sheath gas heater = 325°C; sheath gas flow = 12 L min-1; capillary voltage = 3500 V. Detection of 3HK and 3HAA was by multiple reaction monitoring (MRM) in positive ion mode using the above general mass spectrometry parameters with fragmentor voltage at 380 V and cell accelerator voltage at 5 V. In each case, the fragment ions generated by collision-induced dissociation of the [M+H]+ ion were used for quantification. MRM settings for the target analytes were (parent ion→ fragment ion); 3HK (m/z 225→ 110) with collision energy (CE) = 10 V; 3HAA (m/z 154→ 136) with CE = 9 V. All analytes were quantified against authentic commercial standards obtained from Sigma Aldrich (USA).
Analysis of embryo morphology in mice
[0192] Skeletal morphology was examined according to Wallin et al (Development 1994; 120(5): 1109-21). Heart morphology was examined using optical projection tomography according to Shi et al (Development 2016; 143(14):2561-72.). Morphology of the kidney, palate, and limbs was examined using light microscopy.
DISCUSSION
[0193] For the first time mutations blocking de novo NAD synthesis have been identified as causing multiple congenital malformations, including those found in
VACTERL association. The NAD de novo synthesis pathway catabolizes tryptophan leading to the production of NAD. Although metabolites upstream of the block are elevated and have postnatal functions, it is the deficiency in embryonic NAD,
downstream of the block, that is disrupting embryogenesis. Importantly, supplementing dietary niacin during gestation can prevent embryo defects caused by NAD deficiency. Functional studies establish the damaging effects of truncating variants in the HAAO and KYNU genes, and provide strong evidence for their pathogenicity in multiple congenital malformations. In combination with population data, in silico prediction and variant segregation in the family, the variants are classified "pathogenic" according to the ACMG guidelines (Table 21).
Figure imgf000069_0001
[0194] NAD deficiency is linked to disease via both genetic and environmental means. Severe niacin deficiency causes pellagra characterized by dermatitis, diarrhea, dementia, and death. Although pellagra is rare today, niacin deficiency caused by dietary inadequacies, malabsorption of nutrients and drug interference is still observed. Niacin deficiency can also be inherited; Hartnup disease characterized by dermatitis, neurologic, and behavioral defects is caused by mutation of SLC6A19, required for transport of neutral amino acids including tryptophan. NAD synthesis is also reduced by pathophysiological factors such as type 2 diabetes, obesity, inflammation. These may have been confounding factors in some of the families studied. Niacin deficiency is also common during pregnancy; at least a third of women are deficient in niacin in the first trimester and double this by the third trimester.
[0195] The consequences of the findings presented herein are remarkable as they concern both genetic and environmental causes of congenital malformation. The discovery that genetically disrupting NAD synthesis causes congenital malformations suggests that mutation of many genes might have the same effect (Figure 2). These include genes required for de novo synthesis of NAD, such as tryptophan transporters (SLC7A5, SLC7A8, SLC6A19), enzymes of the kynurenine pathway (TD02, ID01-2, AFMID, KMO, KYNU, HAAO) and enzymes that convert quinolinic acid to NAD (QPRT, NMNAT1-3, NADSYN1). Furthermore, genes encoding enzymes of the NAD salvage pathway (NAMPT, NMNAT1-3), those required for nicotinic acid transport (SLC5A8, SLC22A13), uptake of dietary niacin (nicotinic acid, nicotimamide) or nicotimamide riboside, or for their entry into the NAD producing pathways (NAPRT, PNP, NMRK1 ,2) could also be candidates for causing congenital malformations. It is noteworthy that mutation of NMNAT1 causes the retinal degenerative condition Leber congenital amaurosis type 9 (OMIM 608553) but not congenital malformation. Here, the largely normal enzymatic activity of mutant proteins would preclude a broad phenotype such as congenital malformation.
[0196] The discovery that NAD deficiency can cause congenital malformation is groundbreaking. These findings also predict that interaction between a genetic predisposition to NAD deficiency and a niacin-deficient diet significantly increases the risk of congenital malformation. The use of dietary niacin supplementation to prevent defects could be a simple solution.
[0197] NAD is a co-factor with broad cellular utility involved in ATP production, PARP-dependent-DNA repair and sirtuin-dependent protein deacetylation. NAD+/NADH participates in -400 redox reactions, NADP+/NADPH in -30 redox reactions, and NAD+ is degraded in -50 reactions involving PARP-dependent DNA repair or sirtuin-dependent deacetylation. Given this, there are many possible ways by which an NAD deficit might disrupt embryogenesis.
[0198] First, NAD is central to energy metabolism serving as an essential coenzyme in glycolysis, the tricarboxylic acid cycle, and the mitochondrial electron transport chain. Embryogenesis requires metabolic "fluidity" between glycolysis and oxidative phosphorylation, as stem/progenitor cells transition from proliferation to differentiation. During embryogenesis, these transitions occur in spatially and temporally defined regions, with disruption likely to alter morphogenesis. Moreover, depletion of cellular NAD causes arrest of glycolysis, failure of ATP production, and loss of mitochondrial membrane integrity, resulting in cell apoptosis and necrosis. Since macromolecular synthesis requires ATP, NADPH, and metabolic intermediates derived from glycolysis and TCA cycle, it is conceivable that NAD depletion may also inhibit biomass synthesis in proliferating cells, resulting in growth retardation. Therefore, embryonic regions with the greatest energy demand might be those most affected by NAD deficit. Consistent with this, the heart switches to oxidative phosphorylation early in development and disruption of this could induce heart defects. In addition, loss of ATP production in nephron progenitor cells is associated with hypoplastic kidneys.
[0199] Secondly, NAD levels regulate the deacetylase activity of the sirtuin family of enzymes that modulate activity of transcription factors and metabolic enzymes that promote mitochondrial oxidative function, and enhance cell survival under stress conditions. There is evidence that sirtuin activity is required during embryogenesis for stem/progenitor cell fate decisions, and loss of activity causes multiple organ defects. Therefore an NAD deficit during embryogenesis could lead to defects due to reduced sirtuin activity.
[0200] Thirdly, DNA repair is active during rodent embryogenesis and is required for normal development. DNA base excision repair occurs via poly ADP ribosyl polymerase (PARP) activity, which is responsible for the majority of NAD catabolism. There is evidence that NAD deficiency impairs DNA repair, and that a human DNA fragmentation disorder, Fanconi anemia, can result in multiple congenital malformations defining VACTERL. Taken together, these suggest that specific regions of the embryo require DNA repair and if this is impaired, developmental processes are disrupted.
[0201] In the patients presented, there is a consistency in the tissues affected, but variability in phenotype. Given the phenotypic spectrum and the essential role of NAD there might be even more variable phenotypes that are yet to be discovered. Such variability is likely due to a range of factors including: gene modifiers in both the mother and the affected child; maternal physiology that is influenced, for example by diabetes; diet, including the intake of niacin and tryptophan; and other factors affecting NAD production, salvage and catabolism.
[0202] In conclusion, many genetic and/or environmental factors have the potential to cause NAD deficiency during gestation. Cases of congenital malformation that occur due to an NAD deficit may be collectively referred to as Congenital NAD Deficiency Disorders. The consequences of severe NAD deficiency during gestation. It is conceivable that less extreme NAD deficiency might cause less complex cases of congenital malformation, for example isolated heart defects. High niacin supplementation before and during pregnancy might prevent recurrence of disease in these four families. Niacin supplementation might improve the speech and developmental delay in the surviving patients. Niacin supplementation could reduce the incidence and impact of congenital malformations, more generally.
[0203] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific
embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

Claims:
1. A method of preventing or treating a congenital malformation in an unborn
offspring of a subject, the method comprising administering to the subject an effective amount of NAD or an intermediate of NAD synthesis, wherein the congenital malformation is associated with or caused by at least one of a mutation in a gene encoding an enzyme involved in NAD synthesis;
a dietary deficiency of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid;
inadequate absorption of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid; and
inadequate synthesis of NAD.
2. A method of claim 1 wherein the subject is the mother of an unborn offspring, the unborn offspring or a born offspring.
3. The method of claim 1 or 2 wherein the intermediate of NAD synthesis is selected from the group consisting of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, 2-amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, 2-aminomuconate, 2-oxoadipate, glutaryl-CoA, N- methylnicotinamide (MNA), deamido-NADN-methyl-4-pyridone-3-carboxamide (4- Py), N-methyl-2-pyridone-5-carboxamide (2-Py), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin, vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
4. The method of claim 3 wherein the intermediate of NAD synthesis is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin, vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
5. The method of claim 4 wherein the intermediate is at least one of nicotinamide or niacin.
6. The method of any one of claims 1 to 5 wherein the intermediate of NAD synthesis is administered at a dose of at least about 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 25 mg/kg, 50 mg/kg, at least about 100 mg/kg, at least about 150 mg/kg, or at least about 200 mg/kg body weight of the subject.
7. The method of any one of claims 1 to 6 wherein the NAD or an intermediate of
NAD synthesis is administered orally or by injection.
8. The method of claim 7 wherein the injection is a subcutaneous, intramuscular or intra-venous injection.
9. The method of any one of claims 1 to 8 wherein the NAD or an intermediate of
NAD synthesis is administered before conception, during the term of a pregnancy or after birth.
10. Use of an intermediate of NAD synthesis for the prevention or treatment of a
congenital malformation in a born or unborn offspring of a subject, wherein the congenital malformation is associated with or caused by at least one of:
a mutation in a gene encoding an enzyme involved in NAD synthesis;
a dietary deficiency of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid;
inadequate absorption of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid; and
inadequate synthesis of NAD.
1 1. The use of claim 10 wherein the intermediate of NAD synthesis is selected from the group consisting of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, 2-amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, 2-aminomuconate, 2-oxoadipate, glutaryl-CoA, N-methylnicotinamide (MNA), deamido-NADN-methyl-4-pyridone-3-carboxamide (4-Py), N-methyl-2-pyridone-5- carboxamide (2-Py), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin, vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
12. The use of claim 1 1 wherein the intermediate of NAD synthesis is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin, a derivative thereof, a salt thereof, and any combination thereof.
13. The use of claim 1 1 wherein the intermediate is at least one of vitamin B3,
nicotinamide or niacin.
14. A supplement comprising NAD or an intermediate of NAD synthesis when used for the prevention or treatment of a congenital malformation in a born or unborn offspring of a subject wherein the congenital malformation is associated with or caused by at least one of:
a mutation in a gene encoding an enzyme involved in NAD synthesis;
a dietary deficiency of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid;
inadequate absorption of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid; and
inadequate synthesis of NAD.
15. The supplement of claim 14 wherein the intermediate of NAD synthesis is selected from the group consisting of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, 2-amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, 2-aminomuconate, 2-oxoadipate, glutaryl-CoA, N- methylnicotinamide (MNA), deamido-NADN-methyl-4-pyridone-3-carboxamide (4- Py), N-methyl-2-pyridone-5-carboxamide (2-Py), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin, vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
16. The supplement of claim 15 wherein the intermediate of NAD synthesis is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin, vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
17. The supplement of claim 16 comprising at least one of nicotinamide or niacin. 18. The supplement of any one of claims 14 to 17 wherein a dose of the composition comprises about 10 mg, 15 mg,
18 mg, 20 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 95 Omg, 1 ,000 mg, 2,000 mg or about 3,000 mg of the NAD or an intermediate of NAD synthesis.
19. A method of detecting potential for a congenital malformation in a subject, the method comprising detecting a mutation in a gene encoding an enzyme involved in NAD synthesis from a sample from the subject, wherein the presence of the mutation in the gene is associated with the congenital
malformation.
20. The method of claim 19 wherein the subject is one or both parents, a born
offspring or an unborn offspring.
21. The method of claim 20 wherein the presence of the mutation is indicative of a predisposition to a congenital malformation in the subject's unborn offspring.
22. The method of any one of claims 19 to 21 wherein the congenital malformation is selected from the group consisting of a vertebral defect, sacral agenesis, spinal dysraphism, a cardiac defect, a hypoplastic kidney, a solitary kidney, a shortened long bone; a limb abnormality, submucous cleft palate, bifid uvulua; laryngeal web, vocal cord palsy, anterior anus, restrictive lung disease, microcephaly, and any combination thereof.
23. The method of any one of claims 19 to 22 wherein the gene is selected from the group consisting of aminoadipate aminotransferase, aminocarboxymuconate semialdehyde decarboxylase, acid phosphatase 5, arylformamidase, an alkaline phosphatase, aldehyde oxidase 1 , ADP-ribosyltransferase 1 , ADP- ribosyltransferase 2, ADP-ribosyltransferase 3, ADP-ribosyltransferase 4, ADP- ribosyltransferase 5, bone marrow stromal cell antigen 1 , catalase, CD38, cytochrome B5 reductase 3, cytochrome p450 family 2 subfamily D member 6, cytochrome p450 family 8 subfamily B member 1 , ecto nucleotide
pyrophosphatase/phosphodiesterase 1 , ectonucleotide
pyrophosphatase/phosphodiesterase 2, flavin adenine dinucleotide synthetase 1 , glutamate-ammonia ligase, 3-hydroxyanthranilate 3,4-dioxygenase, indoleamine 2,3-dioxygenase 1 , indoleamine 2,3-dioxygenase 2, kynurenine 3- monooxygenase, kynurenine aminotransferase 1 , kynurenine aminotransferase 3, kynureninase, leukosialin, NAD kinase, NAD kinase 2, NAD synthetase 1 , nicotinamide phosphoribosyltransferase, nicotinate phosphoribosyltransferase, NAD(P)HX dehydratase, NAD(P)HX epimerase, nicotinamide nucleotide adenylyltransferase 1 , nicotinamide nucleotide adenylyltransferase 2, nicotinamide nucleotide adenylyltransferase 3, nicotinamide riboside kinase 1 , nicotinamide riboside kinase 2, nicotinamide N-methyltransferase, nicotinamide nucleotide transhydrogenase, 5', 3'-nucleotidase - cytosolic, 5'-nucleotidase - cytosolic IA, 5'- nucleotidase, cytosolic IB, NT5C1 B-RDH14 readthrough, 5'-nucleotidase - cytosolic I I, 5'-nucleotidase - cytosolic II IA, 5'-nucleotidase - cytosolic NIB, 5'- nucleotidase ecto, 5',3'-nucleotidase - mitochondrial, nudix hydrolase 12, poly(ADP-ribose) polymerase 1 , poly(ADP-ribose) polymerase 2, poly(ADP- ribose) polymerase family member 3, poly(ADP-ribose) polymerase family member 4, poly(ADP-ribose) polymerase family member 6, poly(ADP-ribose) polymerase family member 8, poly(ADP-ribose) polymerase family member 9, poly(ADP-ribose) polymerase family member 10, poly(ADP-ribose) polymerase family member 14, poly(ADP-ribose) polymerase family member 16, pyridoxal (pyridoxine, vitamin B6) kinase, purine nucleoside phosphorylase, pyridoxamine 5'-phosphate oxidase, prostaglandin I2 synthase, prostaglandin-endoperoxide synthase 2, quinolinate phosphoribosyltransferase, riboflavin kinase, sirtuin 1 , sirtuin 2, sirtuin 3, sirtuin 4, sirtuin 5, sirtuin 6, sirtuin 7, solute carrier family 3 member 2, solute carrier family 5 member 8, solute carrier family 6 member 19, solute carrier family 7 member 5, solute carrier family 7 member 8, solute carrier family 16 member 10, solute carrier family 22 member 13, solute carrier family 36 member 4, solute carrier family 52 member 1 , solute carrier family 52 member 2, solute carrier family 52 member 3, tryptophan 2,3-dioxygenase, tankyrase, tankyrase 2, and xanthine dehydrogenase.
24. The method of any one of claims 19 to 22 wherein the gene is selected from the group consisting of a tryptophan transporter, TD02 (tryptophan 2,3-dioxygenase), IDO 1 ,2 (indoleamine 2,3-dioxygenase), AFMID (arylformamidase), KYNB (kynurenine formamidase), KMO (kynurenine 3-monooxygenase), KYNU
(kynureninase), HAAO (3-hydroxyanthranilate 3,4-dioxygenase), ACSMD
(aminocarboxymuconate semialdehyde decarboxylase), QPRT (quinolinate phosphoribosyltransferase), NAPRT, NADSYN1 , NMAT1 , 2, or 3 (nicotinamide nucleotide adenylyltransferase 1 , 2, or 3), NADSYN1 (NAD synthetase 1), nicotinamide phosphoribosyltransferase, NMRK1 ,2 (Nicotinamide riboside kinase 1 or 2), NAMPT (nicotinamide phosphoribosyltransferase), PNP (purine nucleoside phosphorylase), PARP1-6 (poly(ADP-ribose) polymerase 1-6), ART 1- 4, SIRT1-7 (Sirtuin 1-7), CD38 (cyclic ADP ribose hydrolase), BST1 (ADP-ribosyl cyclase 2), TRPT-1 (tRNA 2-phosphotransferase 1), and any combination thereof.
25. The method of claim 24, wherein the mutation is in HAAO or KYNU.
26. The method of claim 25 wherein the HAAO mutation is c.483dupT or c.559G>A.
27. The method of claim 26 wherein the KYNU mutation is c.170-1 G>T,
c.1045_1051delTTTAAGC, or c.468>A.
28. The method of any one of claims 19 to 27 wherein the mutation is detected by nucleic acid sequencing, multiplex ligation dependent probe amplification, single strand conformational polymorphism, or restriction fragment length polymorphism.
29. The method of any one of claims 19 to 28 further comprising administering to the subject or the subject's mother an effective amount a supplement comprising at least one intermediate of NAD synthesis according to any one of claims 14 to 18.
30. A method of detecting potential for a congenital malformation in a subject, the method comprising detecting at least one of:
a. a dietary deficiency of one or more of tryptophan, nicotinamide,
nicotinamide riboside, vitamin B3 and nicotinic acid;
b. inadequate absorption of one or more of tryptophan, nicotinamide, nicotinamide riboside, vitamin B3 and nicotinic acid; and,
c. inadequate synthesis of NAD,
wherein the presence of any one of a, b or c is associated with the congenital malformation.
31. The method of claim 30 wherein the subject is the mother of an unborn offspring or the unborn offspring.
32. The method of claim 31 wherein the presence of any one of a, b or c is indicative of a predisposition to a congenital malformation in the unborn offspring.
33. The method of any one of claims 30 to 32 wherein the congenital malformation is selected from the group consisting of a vertebral defect, sacral agenesis, spinal dysraphism, a cardiac defect, a hypoplastic kidney, a solitary kidney, a shortened long bone; a limb abnormality, submucous cleft palate, bifid uvulua; laryngeal web, vocal cord palsy, anterior anus, restrictive lung disease, microcephaly, and any combination thereof.
34. The method of any one of claims 30 to 33 further comprising administering to the subject or the mother of the subject an effective amount a supplement comprising at least one intermediate of NAD synthesis according to any one of claims 14 to 18.
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WO2020115764A1 (en) * 2018-12-05 2020-06-11 Celagenex Research (India) Pvt. Ltd. Synergistic compositions of bioactive agents for optimizing cellular health
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CN111374310A (en) * 2020-04-16 2020-07-07 上海益跬尔生物科技有限公司 Edible compound mitochondrial energy enhancing formula
CN113893262A (en) * 2021-09-29 2022-01-07 武汉英纽林生物科技有限公司 NAD + composition for intervening high fasting blood glucose in early stage of diabetes as well as preparation method and using method thereof

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