WO2020190608A2 - Treatment of hyperglycemia with solute carrier family 39 member 5 (slc39a5) inhibitors - Google Patents

Treatment of hyperglycemia with solute carrier family 39 member 5 (slc39a5) inhibitors Download PDF

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Publication number
WO2020190608A2
WO2020190608A2 PCT/US2020/022251 US2020022251W WO2020190608A2 WO 2020190608 A2 WO2020190608 A2 WO 2020190608A2 US 2020022251 W US2020022251 W US 2020022251W WO 2020190608 A2 WO2020190608 A2 WO 2020190608A2
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Prior art keywords
seq
slc39a5
nucleic acid
nucleotide sequence
acid molecule
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PCT/US2020/022251
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English (en)
French (fr)
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WO2020190608A3 (en
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Harikiran Nistala
Cristopher Van Hout
Lyndon MITNAUL
Anthony MARCKETTA
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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Priority to EP20717451.7A priority Critical patent/EP3938513A2/en
Priority to CN202080027032.4A priority patent/CN113677368A/zh
Priority to CA3132985A priority patent/CA3132985A1/en
Priority to AU2020241502A priority patent/AU2020241502B2/en
Priority to KR1020217032315A priority patent/KR20210139322A/ko
Priority to MX2021011015A priority patent/MX2021011015A/es
Priority to JP2021555534A priority patent/JP7575396B2/ja
Priority to NZ780568A priority patent/NZ780568A/en
Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Priority to SG11202109711W priority patent/SG11202109711WA/en
Publication of WO2020190608A2 publication Critical patent/WO2020190608A2/en
Publication of WO2020190608A3 publication Critical patent/WO2020190608A3/en
Priority to IL286308A priority patent/IL286308A/en
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Definitions

  • This application includes a Sequence Listing submitted electronically as a text file named 18923802302SEQ, created on March 12, 2020, with a size of 136 kilobytes.
  • the Sequence Listing is incorporated herein by reference.
  • the present disclosure relates generally to the treatment of subjects having hyperglycemia with Solute Carrier Family 39 Member 5 (SLC39A5) inhibitors, methods of identifying subjects having an increased risk of developing hyperglycemia, and methods of detecting SLC39A5 variant nucleic acid molecules and variant polypeptides.
  • Solute Carrier Family 39 Member 5 SLC39A5
  • SLC39A5 belongs to the ZIP (Zrt and Irt-like proteins) superfamily of metal ion transporters. In mice, 14 members of this family have been identified, and almost all of these genes are conserved in humans. ZIP proteins have 8 predicted transmembrane domains (SLC39A5 has 6 transmembrane domains), containing an intracellular loop between transmembrane domains 3 and 4 and an extracellular amino terminus that is histidine-rich and may play a role in metal transport. This zinc transporter localizes to the baso-lateral cell membrane and is expressed in the small intestine, pancreas, kidney and the liver with similar expression pattern in mouse.
  • SLC39A5 is abundant specifically in intestinal enterocytes, pancreatic acinar cells, and embryonic visceral endoderm cells. SLC39A5 regulation also appears to be unique in that this protein is internalized and degraded coordinately in each of these cell-types during periods of dietary zinc deficiency. Translation of the SLC39A5 mRNA is stalled during zinc deficiency in a mechanism which involves a conserved 3'-untranslated region that is predicted to form a stable stem-loop structure and to interact with specific microRNAs. SLC39A4 and SLC39A5 are co-expressed in several tissues with antagonistic functions in maintaining zinc homeostasis.
  • SLC39A4 is thought to facilitate absorption of dietary zinc, whereas SLC39A5 may function in the removal of zinc from the body.
  • Previous studies revealed a role for enterocyte SLC39A5 in zinc excretion, and a role for pancreatic acinar cell SLC39A5 in protection against zinc toxicity.
  • the present disclosure also provides methods of treating a subject having decreased serum zinc level, the methods comprising administering a Solute Carrier Family 39 Member 5 (SLC39A5) inhibitor to the subject.
  • a Solute Carrier Family 39 Member 5 (SLC39A5) inhibitor to the subject.
  • the present disclosure also provides methods of treating a subject having increased serum glucose level, the methods comprising administering an SLC39A5 inhibitor to the subject.
  • the present disclosure also provides methods of treating a subject having
  • hyperglycemia the methods comprising administering an SLC39A5 inhibitor to the subject.
  • the present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits decreased serum zinc level, increased serum glucose level, and/or hyperglycemia wherein the subject is suffering from decreased serum zinc level, increased serum glucose level, and/or hyperglycemia, the methods comprising the steps of: determining whether the subject has an SLC39A5 predicted loss-of-function variant nucleic acid molecule encoding a human SLC39A5 polypeptide by: obtaining or having obtained a biological sample from the subject; and performing or having performed a genotyping assay on the biological sample to determine if the subject has a genotype comprising the SLC39A5 predicted loss-of- function variant nucleic acid molecule; and when the subject is SLC39A5 reference, then administering or continuing to administer to the subject the therapeutic agent that treats or inhibits decreased serum zinc level, increased serum glucose level, and/or hyperglycemia in a standard dosage amount, and administering to the subject an SLC39A5 inhibitor; and when the subject is heterozygous for
  • the present disclosure also provides methods of identifying a human subject having an increased risk for developing decreased serum zinc level, increased serum glucose level, and/or hyperglycemia, wherein the methods comprise: determining or having determined the presence or absence of an SLC39A5 predicted loss-of-function variant nucleic acid molecule encoding a human SLC39A5 polypeptide in a biological sample obtained from the subject; wherein: when the human subject is SLC39A5 reference, then the human subject has an increased risk for developing decreased serum zinc level, increased serum glucose level, and/or hyperglycemia; and when the human subject is heterozygous for an SLC39A5 predicted loss-of- function variant or homozygous for an SLC39A5 predicted loss-of-function variant, then the human subject has a decreased risk for developing decreased serum zinc level, increased serum glucose level, and/or hyperglycemia.
  • the present disclosure also provides methods of detecting a human SLC39A5 variant nucleic acid molecule in a human subject comprising assaying a sample obtained from the human subject to determine whether a nucleic acid molecule in the sample comprises a nucleotide sequence comprising: i) a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, or the complement thereof; ii) a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll, or the complement thereof; iii) a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:18, or the complement thereof; iv) a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6, or the complement thereof; v) a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:13, or the complement thereof; vi) a cytosine at a position
  • the present disclosure also provides methods of detecting the presence of a human SLC39A5 variant polypeptide, comprising performing an assay on a sample obtained from a human subject to determine whether an SLC39A5 protein in the sample: i) comprises a threonine at a position corresponding to position 304 according to SEQ ID NO:24; ii) comprises a alanine at a position corresponding to position 413 according to SEQ ID NO:26; iii) is an SLC39A5 truncated variant polypeptide having an amino acid sequence consisting of SEQ ID NO:23; iv) is an SLC39A5 truncated variant polypeptide having an amino acid sequence consisting of SEQ ID NO:25; or v) is an SLC39A5 truncated variant polypeptide having an amino acid sequence consisting of SEQ ID NO:27.
  • the present disclosure also provides therapeutic agents that treat or inhibit decreased serum zinc level, increased serum glucose level, and/or hyperglycemia for use in the treatment of decreased serum zinc level, increased serum glucose level, and/or hyperglycemia in a human subject having: i) a genomic nucleic acid molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises: a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, or the complement thereof; a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6, or the complement thereof; an adenine or guanine at a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3, or the complement thereof; a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5, or the complement thereof; or a thymine at a position corresponding to position 5,
  • the present disclosure also provides SLC39A5 inhibitors for use in the treatment of decreased serum zinc level, increased serum glucose level, and/or hyperglycemia in a human subject having: i) a genomic nucleic acid molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises: a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, or the complement thereof; a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6, or the complement thereof; an adenine or guanine at a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3, or the complement thereof; a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5, or the complement thereof; or a thymine at a position corresponding to position 5,624 according to SEQ ID NO:7, or the complement thereof;
  • Figure 1A shows SLC39A5 mutant expression in dsRED reporter system.
  • Figure IB shows that SLC39A5 mutations result in loss of function.
  • Figure 2A shows a timeline of experiment testing zinc supplementation rescue of hepatic steatosis and glucose tolerance.
  • Figure 2B shows serum zinc levels in mice following a 4-week high fat diet with or without zinc supplementation.
  • Figure 2C shows hepatic zinc levels in mice following a 4-week high fat diet with or without zinc supplementation.
  • Figure 2D shows serum zinc levels in mice following a 6-week high fat diet with or without zinc supplementation.
  • Figure 2E shows hepatic zinc levels in mice following a 6-week high fat diet with or without zinc supplementation.
  • Figure 3A shows intravenous glucose tolerance test results in mice following a 4-week high fat diet with or without zinc supplementation.
  • Figure 3B shows area under the curve in mice following a 4-week high fat diet with or without zinc supplementation.
  • Figure 3C shows intravenous glucose tolerance test results in mice following a 6-week high fat diet with or without zinc supplementation.
  • Figure 3D shows area under the curve in mice following a 6-week high fat diet.
  • Figure 4 shows micrographs of mouse liver cross-sections following high fat diet with or without zinc supplementation.
  • Figure 5A shows body weights of 12-week old female mice having heterozygous or homozygous knockout of SLC39A5.
  • Figure 5B shows fasting blood glucose levels in 12-week old female mice having heterozygous or homozygous knockout of SLC39A5.
  • Figure 5C shows serum zinc levels in 12-week old male and female mice having heterozygous or homozygous knockout of SLC39A5.
  • Figure 5D shows body weights of 12-week old male mice having heterozygous or homozygous knockout of SLC39A5.
  • Figure 5E shows fasting blood glucose levels in 12-week old male mice having heterozygous or homozygous knockout of SLC39A5.
  • Figure 5F shows hepatic zinc levels in 12-week old male and female mice having heterozygous or homozygous knockout of SLC39A5.
  • Figure 6A shows serum zinc levels after 25 week of high fat diet in 31-week old mice having knockout of SLC39A5.
  • Figure 6B shows fasting blood glucose levels after 25 weeks of high fat diet in 31-week old mice having knockout of SLC39A5.
  • Figure 6C shows leptin levels after 25 weeks of high fat diet in 31-week old mice having knockout of SLC39A5.
  • Figure 6D shows insulin levels after 25 weeks of high fat diet in 31-week old mice having knockout of SLC39A5.
  • Figure 6E shows c-peptide levels after 25 weeks of high fat diet in 31-week old mice having knockout of SLC39A5.
  • Figure 6F shows insulin/c-peptide ratios after 25 weeks of high fat diet in 31-week old mice having knockout of SLC39A5.
  • Figure 7A shows body weights of 12-week old female mice having SLC39A5 knockout, leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • LPR leptin receptor
  • Figure 7B shows fasting blood glucose levels in 12-week old female mice having SLC39A5 knockout, leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • LPR leptin receptor
  • Figure 7C shows serum zinc levels in 12-week old female mice having SLC39A5 knockout , leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • Figure 7D shows body weights of 12-week old male mice having SLC39A5 knockout, leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • LPR leptin receptor
  • Figure 7E shows fasting blood glucose levels in 12-week old male mice having SLC39A5 knockout, leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • Figure 7F shows serum zinc levels in 12-week old male mice having SLC39A5 knockout, leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • Figure 8A shows body weights of 10-week old female mice having SLC39A5 knockout, leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • LPR leptin receptor
  • Figure 8B shows fasting blood glucose levels in 10-week old female mice having SLC39A5 knockout, leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • LPR leptin receptor
  • Figure 8C shows b-hydroxybutyrate levels in 10-week old female mice having SLC39A5 knockout , leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • Figure 8D shows body weights of 10-week old male mice having SLC39A5 knockout, leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • LPR leptin receptor
  • Figure 8E shows fasting blood glucose levels in 10-week old ma le mice having SLC39A5 knockout, leptin receptor (LEPR) knockout, or double SLC39A5/LEPR knockout.
  • LPR leptin receptor
  • Figure 9A shows intravenous glucose tolerance test results in SLC39A5 knockout mice following a 14-week high fat diet.
  • Figure 9B shows insulin blood levels in SLC39A5 knockout mice following a 16-week high fat diet.
  • Figure 10 shows comparative untransformed trait values for various traits in wild type and SLC39A5 knockout mice.
  • Figure 11 shows loss of SLC39A5 results in an increase in serum and bone zinc levels in both sexes, whereas hepatic zinc is modestly elevated only in female mice.
  • Figure 12 shows loss of SLC39A5 improves hepatic steatosis in female mice resulting from diet-induced obesity.
  • Figure 13 shows loss of SLC39A5 improves liver function and certain lipid traits in female mice upon diet-induced obesity challenge.
  • Figure 14 shows loss of SLC39A5 results in reduced hepatic triglyceride content and increased ketogenesis in female mice on a high-fat challenge.
  • FIG. 15 shows SLC39A5 loss-of-function improves liver function in leptin receptor deficient mice.
  • Figure 16 shows SLC39A5 loss-of-function improves glycemic traits in leptin receptor deficient mice.
  • Figure 17 shows loss of SLC39A5 results in hepatic AMP-activated protein kinase activation in female mice on H FD.
  • Figure 18 shows the selection of an SLC39A5 target peptide for mouse immunization from the combined plots of Kyle-Doolitle hydrophobicity (green) and Hopp-Woods
  • FIG 19 shows Slc39a5 monoclonal antibody screening in H EK overexpressing murine
  • FIG. 20 shows Slc39a5 monoclonal antibody screening in H EK overexpressing human
  • Figure 21 shows loss of function of Slc39a5 improves fasting blood glucose upon high fat high fructose dietary challenge.
  • High fat high fructose dietary challenge results in significant increase in body weight across all genotypes in both sexes.
  • Loss of function of Slc39a5 improves liver function as assessed by serum ALT and AST in both sexes at 16 weeks. Loss of function of Slc39a5 significantly improves hyperglycemia assessed by fasting blood glucose levels at endpoint (29 weeks).
  • NC normal chow (NC);
  • HFFD high fat high fructose diet.
  • Figure 22 shows loss of function of Slc39a5 improves insulin sensitivity in mice challenged with high fat high fructose diet. Mice homozygous for Slc39a5 loss of function (regardless of sex) show marked improvement in insulin sensitivity compared to wild type counterparts assessed by oral glucose tolerance tests ("GTT").
  • Figure 23 shows loss of function of Slc39a5 improves hepatic steatosis upon HFFD challenge. Slc39a5 loss of function female mice are more protected than their male
  • NAFLD composite scores (assessed by two independent pathologists) representing an aggregate score of macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis show a significant improvement in female Slc39a5 knockout mice as compared to wild type counterparts; whereas in male mice, loss of Slc39a5 improves hepatic steatosis on normal chow and accords no protection when challenged by high fat high fructose diet demonstrated by histopathology and NAFLD scores.
  • Figures 24A and 24B show loss of function of Slc39a5 results in increased hepatic zinc levels and a consequent elevation in hepatic metallothionein ("Mtl” and "Mt2") expression. Loss of function of Slc39a5 does not significantly influence hepatic iron, copper, cobalt, calcium and magnesium levels (Figure 24A: female mice; Figure 24: male mice).
  • Figures 25A and 25B show zinc acutely activates LKB1/AMPK and AKT signaling pathways in dose dependent manner in human hepatoma HepG2 cells ( Figure 25A) and human primary hepatocytes (Figure 25B).
  • Figure 26A shows loss of function of Slc39a5 improves hepatic steatosis in female mice challenged with high fat high fructose diet.
  • Loss of function of Slc39a5 results in increased hepatic zinc levels with concomitant activation of hepatic AMPK and AKT signaling in female mice challenged with high fat high fructose diet.
  • Hepatic triglyceride levels were reduced with an increase in hepatic beta-hydroxy butyrate levels, suggesting increased b-oxidation.
  • Loss of function of Slc39a5 results in a downregulation of Fasn and G6pc genes involved in de novo lipogenesis and hepatic gluconeogenesis, respectively.
  • Figure 26B shows loss of function of Slc39a5 improves hepatic steatosis in male mice fed with normal chow. Loss of Slc39a5 in male mice challenged with HFFD results in
  • Figure 27 shows loss of function of Slc39a5 improves liver function and fasting blood glucose in leptin-receptor deficient mice.
  • Congenital leptin-receptor deficiency results in significant increase in body weight in Lepr /_ and SIcSgaS ⁇ Lepr ⁇ mice in both sexes.
  • Loss of function of Slc39a5 improves liver function in leptin-receptor deficient mice (both sexes) as assessed by serum ALT and AST at 22 weeks.
  • Loss of function of Slc39a5 significantly improves hyperglycemia in leptin-receptor deficient mice (both sexes) demonstrated by reduced fasting blood glucose levels at 34 weeks.
  • Figure 28 shows loss of function of Slc39a5 in leptin-receptor deficient mice (both sexes) results in improved insulin sensitivity as compared to leptin-receptor deficient
  • Figure 29 shows loss of function of Slc39a5 improves hepatic steatosis in leptin- receptor deficient mice (both sexes).
  • Figure 30 shows loss of function of Slc39a5 results in increased hepatic zinc levels and a consequent elevation in hepatic metallothionein ("Mtl” and "Mt2”) expression in leptin- receptor deficient mice (both sexes). Furthermore, loss function of Slc39a5 does not significantly influence hepatic iron levels in these mice.
  • Figures 31A and 31B show loss of function of Slc39a5 improves hepatic steatosis in leptin receptor deficient mice in both sexes ( Figure 31A, female; Figure 31B, male).
  • Loss of function of Slc39a5 results in increased hepatic zinc levels in leptin receptor deficient mice (both sexes) with concomitant activation of hepatic AMPK signaling.
  • Hepatic triglyceride levels are reduced with an increase in hepatic beta-hydroxybutyrate levels suggesting increased b- oxidation.
  • loss of Slc39a5 results in a downregulation of Fasn indicative of reduced de novo lipogenesis.
  • Figure 32 shows association of loss of function mutations in SLC39A5 with increasing circulating zinc levels and decreased T2D risk (RR: homozygote for reference allele; RA:
  • Figure 33A shows the dependence of tyrosine phosphatase activity on zinc (red line) and magnesium (blue line) concentration.
  • Figure 33B shows the dependence of serine/threonine phosphatase activity on zinc (red line) and magnesium (blue line) concentration.
  • Figure 34A shows that loss of SLC39A5 results in zinc-mediated suppression of tyrosine phosphatase activity in female mice fed with either NC diet (right) or HFFD diet (left).
  • Figure 34B shows that loss of SLC39A5 results in zinc-mediated suppression of serine/threonine phosphatase activity in female mice fed with either NC diet (right) or HFFD diet (left).
  • Figure 34C shows that loss of SLC39A5 results in zinc-mediated suppression of tyrosine phosphatase activity in male mice fed with either NC diet (right) or HFFD diet (left).
  • Figure 34D shows that loss of SLC39A5 results in zinc-mediated suppression of serine/threonine phosphatase activity in male mice fed with either NC diet (right) or HFFD diet (left).
  • the term "subject” includes any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horse, cow, pig), companion animals (such as, for example, dog, cat), laboratory animals (such as, for example, mouse, rat, rabbits), and non-human primates. In some embodiments, the subject is a human.
  • nucleic acid can comprise a polymeric form of nucleotides of any length, can comprise DNA and/or RNA, and can be single-stranded, double-stranded, or multiple stranded.
  • a nucleic acid also refers to its complement.
  • a rare variant in the SLC39A5 gene associated with increased serum zinc levels and a decreased risk of developing increased serum glucose level and/or hyperglycemia, and improved liver function in human subjects has been identified in accordance with the present disclosure.
  • human subjects that are SLC39A5 reference that have an increased risk of developing increased serum glucose level and/or hyperglycemia may be treated such that the increased serum glucose level and/or hyperglycemia is prevented, the symptoms thereof are reduced, and/or development of symptoms is repressed.
  • the present disclosure provides methods of leveraging the identification of such variants in subjects to identify or stratify risk in such subjects of developing increased serum glucose level and/or hyperglycemia, or to diagnose subjects as having an increased risk of developing increased serum glucose level and/or hyperglycemia, such that subjects at risk or subjects with active disease may be treated accordingly.
  • the present disclosure provides isolated SLC39A5 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules. Accordingly, provided herein are SLC39A5 loss-of-function variant nucleic acid molecules discovered to be associated with increased serum zinc levels and a decreased risk of developing increased serum glucose level and/or hyperglycemia.
  • any particular human can be categorized as having one of three SLC39A5 genotypes: i) SLC39A5 reference; ii) heterozygous for an SLC39A5 predicted loss-of-function variant; or iii) homozygous for an SLC39A5 predicted loss-of-function variant.
  • a human is SLC39A5 reference when the human does not have a copy of an SLC39A5 predicted loss-of-function variant nucleic acid molecule.
  • a human is heterozygous for an SLC39A5 predicted loss-of-function variant when the human has a single copy of an SLC39A5 predicted loss-of-function variant nucleic acid molecule.
  • An SLC39A5 predicted loss-of-function variant nucleic acid molecule is any SLC39A5 nucleic acid molecule (such as, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding an SLC39A5 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of- function, or a predicted complete loss-of-function.
  • a human who has an SLC39A5 polypeptide having a partial loss-of-function (or predicted partial loss-of-function) is hypomorphic for SLC39A5.
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule can be any nucleic acid molecule encoding SLC39A5 M304T, G413A, Y47Stop, R322Stop, or R311Stop.
  • the SLC39A5 M304T and G413A are predicted partial loss-of-function variant nucleic acid molecules.
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule encodes SLC39A5 Y47Stop, R322Stop, or R311Stop.
  • a human is homozygous for an SLC39A5 predicted loss-of-function variant when the human has two copies of any of the SLC39A5 predicted loss-of-function variant nucleic acid molecules.
  • human subjects that are genotyped or determined to be SLC39A5 reference such human subjects have an increased risk of developing increased serum glucose level and/or hyperglycemia.
  • human subjects that are genotyped or determined to be either SLC39A5 reference or heterozygous for an SLC39A5 predicted loss-of-function variant such human subjects can be treated with an SLC39A5 inhibitor.
  • such human subjects can be treated with zinc.
  • such human subjects can be treated with a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia.
  • the human subject is a female. In some embodiments, the human subject is a male.
  • the present disclosure provides methods of treating a subject having decreased serum zinc level, the methods comprising administering an SLC39A5 inhibitor to the subject.
  • the present disclosure provides methods of treating a subject having increased serum glucose level, the methods comprising administering an SLC39A5 inhibitor to the subject.
  • the present disclosure provides methods of treating a subject having hyperglycemia, the methods comprising administering an SLC39A5 inhibitor to the subject.
  • the SLC39A5 inhibitor comprises an antibody, or an antigen binding fragment thereof.
  • the antibody specifically binds to a protein comprising SEQ ID NO:22, or a fragment thereof.
  • the antibody specifically binds to a protein comprising SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27, or a fragment thereof.
  • the antibody specifically binds to a peptide comprising the amino acid sequence GPSGWGDQEESKAPDLFIG (SEQ ID NO:67).
  • the antibody comprises a heavy chain comprising the amino acid sequence according to SEQ ID NO:58.
  • the heavy chain is encoded by a
  • the antibody comprises a light chain comprising the amino acid sequence according to SEQ ID NO:60.
  • the light chain is encoded by a
  • the antibody comprises a heavy chain comprising the amino acid sequence according to SEQ ID NO:58, and a light chain comprising the amino acid sequence according to SEQ ID NO:60.
  • the heavy chain of the antibody comprises one or more complementarity-determining regions (CDRs) selected from: FSLTGYAVN (SEQ ID NO:61), WLGVIWGDGRTDY (SEQ ID NO:62), and ARFGNSYALDY (SEQ ID NO:63).
  • the light chain of the antibody comprises one or more CDRs selected from: QSLLNSRTRKNYLA (SEQ ID NO:64), LLIYWASTRES (SEQ ID NO:65), and KQSYN LH (SEQ ID NO:66).
  • the antibody fragment may comprise a single chain (scFv), diabodies, Fv, and (Fab') 2 , triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDR's, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, and the like.
  • the SLC39A5 inhibitor comprises an antisense molecule.
  • antisense molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs). Such antisense molecules can be designed to target any region of an SLC39A5 mRNA. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within an SLC39A5 genomic nucleic acid molecule or mRNA molecule and decreases expression of the SLC39A5 polypeptide in a cell in the subject.
  • siRNAs small interfering RNAs
  • shRNAs short hairpin RNAs
  • the SLC39A5 inhibitor comprises an antisense RNA that hybridizes to an SLC39A5 genomic nucleic acid molecule or mRNA molecule and decreases expression of the SLC39A5 polypeptide in a cell in the subject.
  • the SLC39A5 inhibitor comprises an siRNA that hybridizes to an SLC39A5 genomic nucleic acid molecule or mRNA molecule and decreases expression of the SLC39A5 polypeptide in a cell in the subject.
  • the SLC39A5 inhibitor comprises an shRNA that hybridizes to an SLC39A5 genomic nucleic acid molecule or mRNA molecule and decreases expression of the SLC39A5 polypeptide in a cell in the subject.
  • the SLC39A5 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks at a recognition sequence(s) or a DNA-binding protein that binds to a recognition sequence within an SLC39A5 genomic nucleic acid molecule.
  • the recognition sequence can be located within a coding region of the SLC39A5 gene, or within regulatory regions that influence the expression of the gene.
  • a recognition sequence of the DNA-binding protein or nuclease agent can be located in an intron, an exon, a promoter, an enhancer, a regulatory region, or any non-protein coding region.
  • the recognition sequence can include or be proximate to the start codon of the SLC39A5 gene.
  • the recognition sequence can be located from about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the start codon.
  • two or more nuclease agents can be used, each targeting a nuclease recognition sequence including or proximate to the start codon.
  • two nuclease agents can be used, one targeting a nuclease recognition sequence including or proximate to the start codon, and one targeting a nuclease recognition sequence including or proximate to the stop codon, wherein cleavage by the nuclease agents can result in deletion of the coding region between the two nuclease recognition sequences.
  • nuclease agent that induces a nick or double-strand break into a desired recognition sequence
  • Any DNA-binding protein that binds to a desired recognition sequence can be used in the methods and compositions disclosed herein.
  • Suitable nuclease agents and DNA-binding proteins for use herein include, but are not limited to, zinc finger protein or zinc finger nuclease (ZFN) pair, Transcription Activator-Like Effector (TALE) protein or Transcription Activator-Like Effector Nuclease (TALEN), or Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems.
  • ZFN zinc finger protein or zinc finger nuclease
  • TALE Transcription Activator-Like Effector
  • TALEN Transcription Activator-Like Effector Nuclease
  • CRISPR Clustered Regularly Interspersed Short Palindromic Repeats
  • Cas Clustered Regularly Interspersed Short Palindromic Repeats
  • the length of the recognition sequence can vary, and includes, for example, recognition sequences that are about 30-36 bp for a zinc finger protein or ZFN pair (i.e., about 15-18 bp for each ZFN), about 36 bp for a TALE protein or TALEN, and about 20 bp for a CRISPR/Cas guide RNA.
  • CRISPR/Cas systems can be used to modify an SLC39A5 genomic nucleic acid molecule within a cell.
  • the methods and compositions disclosed herein can employ CRISPR-Cas systems by utilizing CRISPR complexes (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-directed cleavage of SLC39A5 nucleic acid molecules.
  • CRISPR complexes comprising a guide RNA (gRNA) complexed with a Cas protein
  • Cas proteins generally comprise at least one RNA recognition or binding domain that can interact with gRNAs. Cas proteins can also comprise nuclease domains (such as, for example, DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, a wild type Cas9 protein and a wild type Cpfl protein (such as, for example, FnCpfl). A Cas protein can have full cleavage activity to create a double-strand break in an SLC39A5 genomic nucleic acid molecule or it can be a nickase that creates a single-strand break in an SLC39A5 genomic nucleic acid molecule.
  • Cas proteins include, but are not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl
  • Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins.
  • a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • Cas proteins can be provided in any form.
  • a Cas protein can be provided in the form of a protein, such as a Cas protein complexed with a gRNA.
  • a Cas protein can be provided in the form of a nucleic acid molecule encoding the Cas protein, such as an RNA or DNA.
  • targeted genetic modifications of SLC39A5 genomic nucleic acid molecules can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus in the SLC39A5 genomic nucleic acid molecule.
  • a gRNA recognition sequence can be located within a region of SEQ ID NO:l.
  • the gRNA recognition sequence can also include or be proximate to a position corresponding to position i) 5,604, ii) 6,899, iii) 1,353, iv) 6,352, or v) 5,624 according to SEQ ID NO:l.
  • the gRNA recognition sequence can be located from about 1000, 500, 400, 300, 200, 100, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 nucleotides of a position corresponding to position i) 5,604, ii) 6,899, iii) 1,353, iv) 6,352, or v) 5,624 according to SEQ ID NO:l.
  • the gRNA recognition sequence can include or be proximate to the start codon of an SLC39A5 genomic nucleic acid molecule or the stop codon of an SLC39A5 genomic nucleic acid molecule.
  • the gRNA recognition sequence can be located from about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the start codon or the stop codon.
  • the gRNA recognition sequences within a target genomic locus in an SLC39A5 genomic nucleic acid molecule are located near a Protospacer Adjacent Motif (PAM) sequence, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease.
  • the canonical PAM is the sequence 5'-NGG-3' where "N" is any nucleobase followed by two guanine ("G”) nucleobases.
  • gRNAs can transport Cas9 to anywhere in the genome for gene editing, but no editing can occur at any site other than one at which Cas9 recognizes PAM.
  • 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells.
  • the PAM is about 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA.
  • the PAM can flank the gRNA recognition sequence.
  • the gRNA recognition sequence can be flanked on the 3' end by the PAM.
  • the gRNA recognition sequence can be flanked on the 5' end by the PAM.
  • the cleavage site of Cas proteins can be about 1 to about 10, about 2 to about 5 base pairs, or three base pairs upstream or downstream of the PAM sequence.
  • the PAM sequence of the non-lived by the PAM can flank the gRNA recognition sequence.
  • the gRNA recognition sequence can be flanked on the 3' end by the PAM.
  • the gRNA recognition sequence can be flanked on the 5' end by the PAM.
  • the cleavage site of Cas proteins can be about 1 to about 10, about 2 to about 5 base pairs, or three base pairs upstream or downstream of the PAM sequence.
  • complementary strand can be 5'-NGG-3’, where N is any DNA nucleotide and is immediately 3' of the gRNA recognition sequence of the non-complementary strand of the target DNA.
  • PAM sequence of the complementary strand would be 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' of the gRNA recognition sequence of the complementary strand of the target DNA.
  • a gRNA is an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific location within an SLC39A5 genomic nucleic acid molecule.
  • An exemplary gRNA is a gRNA effective to direct a Cas enzyme to bind to or cleave an SLC39A5 genomic nucleic acid molecule, wherein the gRNA comprises a DNA-targeting segment that hybridizes to a gRNA recognition sequence within the SLC39A5 genomic nucleic acid molecule that includes or is proximate to a position corresponding to position i) 5,604, ii) 6,899, iii) 1,353, iv) 6,352, or v) 5,624 according to SEQ ID NO:l.
  • a gRNA can be selected such that it hybridizes to a gRNA recognition sequence that is located from about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
  • exemplary gRNAs comprise a DNA-targeting segment that hybridizes to a gRNA recognition sequence within an SLC39A5 genomic nucleic acid molecule that includes or is proximate to the start codon or the stop codon.
  • a gRNA can be selected such that it hybridizes to a gRNA recognition sequence that is located from about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400,
  • gRNAs are described in, for example, Mali et al., Science, 2013, 339, 823-826; Jinek et al., Science, 2012, 337, 816-821; Flwang et al., Nat. Biotechnol., 2013, 31, 227- 229; Jiang et al., Nat. Biotechnol., 2013, 31, 233-239; and Cong et al., Science, 2013, 339, 819- 823.
  • Suitable gRNAs can comprise from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides. In some embodiments, the gRNAs can comprise 20 nucleotides.
  • gRNA recognition sequences located within the human SLC39A5 reference gene are set forth in Table 1 as SEQ ID NOS:28-56.
  • the Cas protein and the gRNA form a complex, and the Cas protein cleaves the target SLC39A5 genomic nucleic acid molecule.
  • the Cas protein can cleave the nucleic acid molecule at a site within or outside of the nucleic acid sequence present in the target SLC39A5 genomic nucleic acid molecule to which the DNA-targeting segment of a gRNA will bind.
  • formation of a CRISPR complex (comprising a gRNA hybridized to a gRNA recognition sequence and complexed with a Cas protein) can result in cleavage of one or both strands in or near (such as, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the nucleic acid sequence present in the SLC39A5 genomic nucleic acid molecule to which a DNA-targeting segment of a gRNA will bind.
  • Such methods can result, for example, in an SLC39A5 genomic nucleic acid molecule in which a region of SEQ ID NO:l is disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted.
  • the cell can be further contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within the target genomic locus in the SLC39A5 genomic nucleic acid molecule.
  • additional gRNAs such as, for example, a second gRNA that hybridizes to a second gRNA recognition sequence
  • cleavage by the Cas protein can create two or more double-strand breaks or two or more single-strand breaks.
  • the treatment methods further comprise detecting the presence or absence of an SLC39A5 predicted loss-of-function variant nucleic acid molecule encoding a human SLC39A5 polypeptide in a biological sample from the subject.
  • a "SLC39A5 predicted loss-of-function variant nucleic acid molecule” is any SLC39A5 nucleic acid molecule (such as, for example, genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding an SLC39A5 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function.
  • the present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia, wherein the subject is suffering from increased serum glucose level and/or hyperglycemia, the method comprising the steps of: determining whether the subject has an SLC39A5 predicted loss-of- function variant nucleic acid molecule encoding a human SLC39A5 polypeptide by: obtaining or having obtained a biological sample from the subject; and performing or having performed a genotyping assay on the biological sample to determine if the subject has a genotype comprising the SLC39A5 predicted loss-of-function variant nucleic acid molecule; and when the subject is SLC39A5 reference, then administering or continuing to administer to the subject the therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia in a standard dosage amount, and administering to the subject an SLC39A5 inhibitor; and when the subject is heterozygous for an SLC39A5 predicted loss-of-function variant, then
  • the subject is SLC39A5 reference. In some embodiments, the subject is heterozygous for an SLC39A5 predicted loss-of-function variant.
  • human subjects that are genotyped or determined to be either SLC39A5 reference or heterozygous for an SLC39A5 predicted loss-of-function variant, such human subjects can be treated with an SLC39A5 inhibitor, as described herein.
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule can be any SLC39A5 nucleic acid molecule (such as, for example, genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding an SLC39A5 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function.
  • SLC39A5 nucleic acid molecule such as, for example, genomic nucleic acid molecule, mRNA molecule, or cDNA molecule
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule can be any nucleic acid molecule encoding any of the SLC39A5 polypeptides described herein including, for example, SLC39A5 M304T, G413A, Y47Stop, R322Stop, or R311Stop.
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule can also be any SLC39A5 nucleic acid molecule (such as, for example, genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that comprises: i) a point mutation in an exon of a reference SLC39A5 gene; ii) a deletion, in whole or in part, of the coding sequence of a reference SLC39A5 gene, such as, for example, a deletion that comprises exon 1, in whole or in part, and/or exon 2, in whole or in part.
  • SLC39A5 nucleic acid molecule such as, for example, genomic nucleic acid molecule, mRNA molecule, or cDNA molecule
  • a deletion, in whole or in part, of the coding sequence of a reference SLC39A5 gene such as, for example, a deletion that comprises exon 1, in whole or in part, and/or exon 2, in whole or in part.
  • the deletion comprises a coding portion of exon 1 and a portion of exon 2 of a reference SLC39A5 gene. In some embodiments, the deletion comprises a nucleic acid sequence from the nucleotide after the ATG start codon in exon 1 through the fifth nucleotide before the 3' end of exon 2 of a reference SLC39A5 gene.
  • Detecting the presence or absence of an SLC39A5 predicted loss-of-function variant nucleic acid molecule in a biological sample from a subject and/or determining whether a subject has an SLC39A5 predicted loss-of-function variant nucleic acid molecule can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the nucleic acid molecule can be present within a cell obtained from the human subject.
  • the subject when the subject is SLC39A5 reference, the subject is also administered a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia in a standard dosage amount. In some embodiments, when the subject is heterozygous for an SLC39A5 predicted loss-of-function variant, the subject is also administered a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia in a standard dosage amount. In some embodiments, when the subject is heterozygous for an SLC39A5 predicted loss-of-function variant, the subject is also
  • a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia in a dosage amount that is the same as or lower than the standard dosage amount.
  • the treatment methods further comprise detecting the presence or absence of an SLC39A5 predicted loss-of-function polypeptide in a biological sample from the subject.
  • the subject when the subject does not have an SLC39A5 predicted loss-of-function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia in a standard dosage amount.
  • the subject when the subject has an SLC39A5 predicted loss-of-function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia in a dosage amount that is the same as or lower than the standard dosage amount.
  • the present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia, wherein the subject is suffering from increased serum glucose level and/or hyperglycemia, the method comprising the steps of: determining whether the subject has an SLC39A5 predicted loss-of- function polypeptide by: obtaining or having obtained a biological sample from the subject; and performing or having performed an assay on the biological sample to determine if the subject has an SLC39A5 predicted loss-of-function polypeptide; and when the subject does not have an SLC39A5 predicted loss-of-function polypeptide, then administering or continuing to administer to the subject the therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia in a standard dosage amount, and administering to the subject an SLC39A5 inhibitor; and when the subject has an SLC39A5 predicted loss-of-function polypeptide, then administering or continuing to administer to the subject the therapeutic agent that treats or inhibits increased serum glucose level and/or hyper
  • the subject has an SLC39A5 predicted loss-of-function polypeptide. In some embodiments, the subject does not have an SLC39A5 predicted loss-of- function polypeptide.
  • the SLC39A5 predicted loss-of-function polypeptide can be any SLC39A5 polypeptide having a partial loss-of-function, a complete loss- of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function.
  • the SLC39A5 predicted loss-of-function polypeptide can be any of the SLC39A5 polypeptides described herein including, for example, SLC39A5 M304T, G413A, Y47Stop, R322Stop, or R311Stop.
  • the SLC39A5 polypeptide is SLC39A5 Y47Stop, R322Stop, or R311Stop.
  • Detecting the presence or absence of an SLC39A5 predicted loss-of-function polypeptide in a biological sample from a subject and/or determining whether a subject has an SLC39A5 predicted loss-of-function polypeptide can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the polypeptide can be present within a cell obtained from the human subject.
  • the subject may have decreased serum zinc level or decreased bone zinc level, or both.
  • the subject may have any one or more of the following: increased serum glucose level, hyperglycemia, Type 2 diabetes, steroid-induced diabetes, increased low density lipoprotein (LDL), and decreased high density lipoprotein (HDL).
  • the subject may have increased serum glucose level and may be treated by the methods described herein.
  • the subject may have hyperglycemia and may be treated by the methods described herein.
  • the subject may have hyperglycemia and may be treated by the methods described herein.
  • the subject may have steroid-induced diabetes and may be treated by the methods described herein.
  • the subject may have increased LDL and may be treated by the methods described herein.
  • the subject may have decreased HDL and may be treated by the methods described herein.
  • the subject may have Type 2 diabetes and may be treated by the methods described herein.
  • the subject may have any one or more of increased serum glucose level, hyperglycemia, Type 2 diabetes, steroid-induced diabetes, increased LDL, and decreased HDL, with or without concomitant liver dysfunction.
  • Liver dysfunction includes any one or more of the following: fatty liver disease (including alcoholic fatty liver disease (AFLD) and non-alcoholic fatty liver disease (NAFLD)), steatohepatitis, hepatic encephalopathy, liver fibrosis, increased liver enzymes (including alanine transaminase (ALT) and aspartate transaminase (AST)), and iron overload diseases such as hemochromatosis.
  • the subject may have any one or more of increased serum glucose level, hyperglycemia, Type 2 diabetes, steroid-induced diabetes, increased LDL, and decreased HDL, with or without decreased serum zinc level or decreased bone zinc level, or both.
  • Statins include, but are not limited to: atorvastatin, pravastatin, fluvastatin, lovastatin, simvastatin, and cerivastatin.
  • PPAR agonists include thiazolidinediones and fibrates.
  • Thiazolidinediones include, but are not limited to: 5-((4-(2-(methyl-2-pyridinylamino)ethoxy)phenyl)methyl)-2,4- thiazolidinedione, troglitazone, pioglitazone, ciglitazone, WAY-120,744, englitazone, AD 5075, darglitazone, and rosiglitazone.
  • Fibrates include, but are not limited to: gemfibrozil, fenofibrate, clofibrate, or ciprofibrate.
  • Hormones include, but are not limited to: thyroid hormone, estrogen, and insulin. Suitable insulins include, but are not limited to: injectable insulin, transdermal insulin, inhaled insulin, or any combination thereof. As an alternative to insulin, an insulin derivative, secretagogue, sensitizer or mimetic may be used. Insulin secretagogues include, but are not limited to: forskolin, dibutryl cAM P or isobutylmethylxanthine (IBMX).
  • IBMX isobutylmethylxanthine
  • Sulfonylurea-based agents include, but are not limited to: glisoxepid, glyburide, acetohexamide, chlorpropamide, glibornuride, tolbutamide, tolazamide, glipizide, gliclazide, gliquidone, glyhexamide, phenbutamide, and tolcyclamide.
  • Biguanides include, but are not limited to: metformin, phenformin, and buformin.
  • a-glucosidase inhibitors include, but are not limited to: acarbose and miglitol.
  • GLP-1 agonists include, but are not limited to: VICTOZA ® or SAXENDA ® (liraglutide), BYETTA ® or BYDUREON ® (exenatide), LYXUMIA ® (lixisenatide), TANZEUM ® (albiglutide), TRULICITY ® (dulaglutide), and OZEMPIC ® (semaglutide).
  • the dose of the therapeutic agents that treat or inhibit increased serum glucose level and/or hyperglycemia can be reduced by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, or by about 90% for subjects that are heterozygous for an SLC39A5 predicted loss-of-function variant (i.e., a lower than the standard dosage amount) compared to subjects that are SLC39A5 reference (who may receive a standard dosage amount).
  • the dose of the therapeutic agents that treat or inhibit increased serum glucose level and/or hyperglycemia can be reduced by about 10%, by about 20%, by about 30%, by about 40%, or by about 50%.
  • the dose of therapeutic agents that treat or inhibit increased serum glucose level and/or hyperglycemia in subjects that are heterozygous for an SLC39A5 predicted loss-of- function variant can be administered less frequently compared to subjects that are SLC39A5 reference.
  • Administration of the therapeutic agents that treat or inhibit increased serum glucose level and/or hyperglycemia and/or SLC39A5 inhibitors can be repeated, for example, after one day, two days, three days, five days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, eight weeks, two months, or three months.
  • the repeated administration can be at the same dose or at a different dose.
  • the administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more.
  • a subject can receive therapy for a prolonged period of time such as, for example, 6 months, 1 year, or more.
  • Administration of the therapeutic agents that treat or inhibit increased serum glucose level and/or hyperglycemia and/or SLC39A5 inhibitors can occur by any suitable route including, but not limited to, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular.
  • Pharmaceutical compositions for administration are desirably sterile and substantially isotonic and
  • compositions can be provided in unit dosage form (i.e., the dosage for a single administration).
  • Pharmaceutical compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen.
  • pharmaceutically acceptable means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.
  • the human subject can be administered zinc.
  • Zinc can be administered orally as a zinc salt such as, for example, zinc sulfate or zinc gluconate.
  • Zinc can be administered in an amount from about 5 to about 100 mg/day, from about 5 to about 80 mg/day, from about 5 to about 60 mg/day, from about 5 to about 50 mg/day, from about 5 to about 40 mg/day, from about 5 to about 25 mg/day, from about 5 to about 15 mg/day, from about 10 to about 100 mg/day, from about 15 to about 100 mg/day, from about 20 to about 100 mg/day, from about 25 to about 100 mg/day, from about 10 to about 50 mg/day, from about 15 to about 40 mg/day, or from about 20 to about 30 mg/day.
  • a therapeutic effect comprises one or more of a decrease/reduction in increased serum glucose level and/or hyperglycemia, a decrease/reduction in the severity of increased serum glucose level and/or hyperglycemia (such as, for example, a reduction or inhibition of development of increased serum glucose level and/or hyperglycemia), a decrease/reduction in symptoms and increased serum glucose level- and/or hyperglycemia-related effects, delaying the onset of symptoms and increased serum glucose level- and/or hyperglycemia-related effects, reducing the severity of symptoms of increased serum glucose level- and/or hyperglycemia-related effects, reducing the severity of an acute episode, reducing the number of symptoms and increased serum glucose level- and/or hyperglycemia-related effects, reducing the latency of symptoms
  • a prophylactic effect may comprise a complete or partial avoidance/inhibition or a delay of increased serum glucose level and/or hyperglycemia development/progression (such as, for example, a complete or partial avoidance/inhibition or a delay), and an increased survival time of the affected host animal, following administration of a therapeutic protocol.
  • Treatment of increased serum glucose level and/or hyperglycemia encompasses the treatment of subjects already diagnosed as having any form of increased serum glucose level and/or hyperglycemia at any clinical stage or manifestation, the delay of the onset or evolution or aggravation or deterioration of the symptoms or signs of increased serum glucose level and/or hyperglycemia, and/or preventing and/or reducing the severity of increased serum glucose level and/or hyperglycemia.
  • the present disclosure also provides methods of identifying a human subject having an increased risk for developing increased serum glucose level and/or hyperglycemia, wherein the method comprises: determining or having determined in a biological sample obtained from the subject the presence or absence of an SLC39A5 predicted loss-of-function variant nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, and/or cDNA molecule) encoding a human SLC39A5 polypeptide; wherein: i) when the human subject lacks an SLC39A5 predicted loss-of-function variant nucleic acid molecule (i.e., the human subject is genotypically categorized as an SLC39A5 reference), then the human subject has an increased risk for developing increased serum glucose level and/or hyperglycemia; and ii) when the human subject has an SLC39A5 predicted loss-of-function variant nucleic acid molecule (i.e., the human subject is categorized as heterozygous for an SLC39A5 predicted loss-of-
  • Having a single copy of an SLC39A5 predicted loss-of-function variant nucleic acid molecule may be more protective of a human subject from developing increased serum glucose level and/or hyperglycemia than having no copies of an SLC39A5 predicted loss-of-function variant nucleic acid molecule.
  • an SLC39A5 predicted loss-of-function variant nucleic acid molecule i.e., heterozygous for an SLC39A5 predicted loss-of-function variant
  • having two copies of an SLC39A5 predicted loss-of-function variant nucleic acid molecule i.e., homozygous for an SLC39A5 predicted loss-of-function variant
  • a single copy of an SLC39A5 predicted loss-of-function variant nucleic acid molecule may not be completely protective, but instead, may be partially or incompletely protective of a human subject from developing increased serum glucose level and/or hyperglycemia. While not desiring to be bound by any particular theory, there may be additional factors or molecules involved in the development of increased serum glucose level and/or hyperglycemia that are still present in a human subject having a single copy of an SLC39A5 predicted loss-of-function variant nucleic acid molecule, thus resulting in less than complete protection from the development of increased serum glucose level and/or hyperglycemia.
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule can be any SLC39A5 nucleic acid molecule (such as, for example, genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding an SLC39A5 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function.
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule can be any nucleic acid molecule encoding SLC39A5 M304T, G413A, Y47Stop, R322Stop, or R311Stop.
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule encodes SLC39A5 Y47Stop, R322Stop, or R311Stop.
  • Determining whether a human subject has an SLC39A5 predicted loss-of-function variant nucleic acid molecule in a biological sample can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the nucleic acid molecule can be present within a cell obtained from the human subject. ln any of the embodiments described herein, the subject may have decreased serum zinc level or decreased bone zinc level, or both.
  • the subject may have any one or more of the following: increased serum glucose level, hyperglycemia, Type 2 diabetes, steroid-induced diabetes, increased low density lipoprotein (LDL), and decreased high density lipoprotein (HDL).
  • the subject may have increased serum glucose level and may be treated by the methods described herein.
  • the subject may have hyperglycemia and may be treated by the methods described herein.
  • the subject may have steroid-induced diabetes and may be treated by the methods described herein.
  • the subject may have increased LDL and may be treated by the methods described herein.
  • the subject may have decreased HDL and may be treated by the methods described herein.
  • the subject may have Type 2 diabetes and may be treated by the methods described herein.
  • the subject may have any one or more of increased serum glucose level, hyperglycemia, Type 2 diabetes, steroid-induced diabetes, increased LDL, and decreased HDL, with or without concomitant liver dysfunction.
  • Liver dysfunction includes any one or more of the following: fatty liver disease (including alcoholic fatty liver disease (AFLD) and non-alcoholic fatty liver disease (NAFLD)), steatohepatitis, hepatic encephalopathy, liver fibrosis, increased liver enzymes (including alanine transaminase (ALT) and aspartate transaminase (AST)), and iron overload diseases such as hemochromatosis.
  • the subject may have any one or more of increased serum glucose level, hyperglycemia, Type 2 diabetes, steroid-induced diabetes, increased LDL, and decreased HDL, with or without decreased serum zinc level or decreased bone zinc level, or both.
  • the human subject when a human subject is identified as having an increased risk of developing increased serum glucose level and/or hyperglycemia, the human subject is further treated with a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia and/or an SLC39A5 inhibitor, as described herein.
  • a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia and/or an SLC39A5 inhibitor, as described herein.
  • the human subject when the human subject is SLC39A5 reference, and therefore has an increased risk for developing increased serum glucose level and/or hyperglycemia, the human subject is administered an SLC39A5 inhibitor.
  • such a subject is also administered a therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia.
  • the subject when the subject is heterozygous for an SLC39A5 predicted loss-of-function variant, the subject is administered the therapeutic agent that treats or inhibits increased serum glucose level and/or hyperglycemia in a dosage amount that is the same as or lower than the standard dosage amount, and is also administered an SLC39A5 inhibitor.
  • the subject is SLC39A5 reference.
  • the subject is heterozygous for an SLC39A5 predicted loss-of-function variant.
  • the present disclosure also provides methods of detecting the presence of an SLC39A5 predicted loss-of-function variant genomic nucleic acid molecule, an SLC39A5 predicted loss-of- function variant mRNA molecule, and/or an SLC39A5 predicted loss-of-function variant cDNA molecule in a biological sample from a subject human. It is understood that gene sequences within a population and mRNA molecules encoded by such genes can vary due to
  • polymorphisms such as single-nucleotide polymorphisms.
  • sequences provided herein for the SLC39A5 variant genomic nucleic acid molecule, SLC39A5 variant mRNA molecule, and SLC39A5 variant cDNA molecule are only exemplary sequences. Other sequences for the SLC39A5 variant genomic nucleic acid molecule, variant mRNA molecule, and variant cDNA molecule are also possible.
  • the biological sample can be derived from any cell, tissue, or biological fluid from the subject.
  • the sample may comprise any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of bodily fluid, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascitic fluid, cystic fluid, or urine.
  • the sample comprises a buccal swab.
  • the sample used in the methods disclosed herein will vary based on the assay format, nature of the detection method, and the tissues, cells, or extracts that are used as the sample.
  • a biological sample can be processed differently depending on the assay being employed.
  • any SLC39A5 variant nucleic acid molecule when detecting any SLC39A5 variant nucleic acid molecule, preliminary processing designed to isolate or enrich the sample for the genomic DNA can be employed. A variety of known techniques may be used for this purpose. When detecting the level of any SLC39A5 variant mRNA, different techniques can be used enrich the biological sample with mRNA. Various methods to detect the presence or level of a mRNA or the presence of a particular variant genomic DNA locus can be used.
  • detecting a human SLC39A5 predicted loss-of-function variant nucleic acid molecule in a human subject comprises assaying or genotyping a biological sample obtained from the human subject to determine whether an SLC39A5 genomic nucleic acid molecule, an SLC39A5 mRNA molecule, or an SLC39A5 cDNA molecule in the biological sample comprises one or more variations that cause a loss-of-function (partial or complete) or are predicted to cause a loss-of-function (partial or complete).
  • the methods of detecting the presence or absence of an SLC39A5 predicted loss-of-function variant nucleic acid molecule comprise: performing an assay on a biological sample obtained from the human subject, which assay determines whether a nucleic acid molecule in the biological sample comprises a particular nucleotide sequence.
  • the nucleotide sequence comprises: i) a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4 (for genomic nucleic acid molecules); ii) a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll (for mRNA molecules); or iii) a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:18 (for cDNA molecules obtained from mRNA molecules).
  • the nucleotide sequence comprises: i) a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6 (for genomic nucleic acid molecules); ii) a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:13 (for mRNA molecules); or iii) a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:20 (for cDNA molecules obtained from mRNA molecules).
  • the nucleotide sequence comprises: i) an adenine or guanine at a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3 (for genomic nucleic acid molecules); ii) an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:9 or SEQ ID NO:10 (for mRNA molecules); or iii) an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:16 or SEQ ID NO:17 (for cDNA molecules produced from mRNA molecules).
  • the nucleotide sequence comprises: i) a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5 (for genomic nucleic acid molecules); ii) a uracil at a position corresponding to position 1,194 according to SEQ ID NO:12 (for mRNA molecules); or iii) a thymine at a position corresponding to position 1,194 according to SEQ ID NO:19 (for cDNA molecules produced from mRNA molecules).
  • the nucleotide sequence comprises: i) a thymine at a position corresponding to position 5,624 according to SEQ ID NO:7 (for genomic nucleic acid molecules); ii) a uracil at a position corresponding to position 1,161 according to SEQ ID NO:14 (for mRNA molecules); or iii) a thymine at a position corresponding to position 1,161 according to SEQ ID NO:21 (for cDNA molecules produced from mRNA molecules).
  • the biological sample comprises a cell or cell lysate.
  • Such methods can further comprise, for example, obtaining a biological sample from the subject comprising an SLC39A5 genomic nucleic acid molecule or mRNA molecule, and if mRNA, optionally reverse transcribing the mRNA into cDNA.
  • Such assays can comprise, for example determining the identity of these positions of the particular SLC39A5 nucleic acid molecule.
  • the method is an in vitro method.
  • the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the SLC39A5 genomic nucleic acid molecule, the SLC39A5 mRNA molecule, or the SLC39A5 cDNA molecule in the biological sample, wherein the sequenced portion comprises one or more variations that cause a loss-of-function (partial or complete) or are predicted to cause a loss-of-function (partial or complete).
  • the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of: i) the nucleotide sequence of the SLC39A5 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 5,604 according to SEQ ID NO:4, or the complement thereof; ii) the nucleotide sequence of the SLC39A5 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 1,141 according to SEQ ID NO:ll, or the complement thereof; and/or iii) the nucleotide sequence of the SLC39A5 cDNA molecule produced from the mRNA in the biological sample, wherein the sequenced portion comprises a position corresponding to position 1,141 according to SEQ ID NO:18, or the complement thereof.
  • the sequenced portion of the SLC39A5 nucleic acid molecule in the biological sample comprises: a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll, or a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:18
  • the SLC39A5 nucleic acid molecule in the biological sample is an SLC39A5 predicted loss- of-function variant nucleic acid molecule.
  • the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of: i) the nucleotide sequence of the SLC39A5 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 6,899 according to SEQ ID NO:6, or the complement thereof; ii) the nucleotide sequence of the SLC39A5 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 1,468 according to SEQ ID NO:13, or the complement thereof; and/or iii) the nucleotide sequence of the SLC39A5 cDNA molecule produced from the mRNA in the biological sample, wherein the sequenced portion comprises a position corresponding to position 1,468 according to SEQ ID NO:20, or the complement thereof.
  • the sequenced portion of the SLC39A5 nucleic acid molecule in the biological sample comprises: a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6, a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:13, or a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:20, then the SLC39A5 nucleic acid molecule in the biological sample is an SLC39A5 predicted loss- of-function variant nucleic acid molecule.
  • the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of: i) the nucleotide sequence of the SLC39A5 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3, or the complement thereof; ii) the nucleotide sequence of the SLC39A5 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 371 according to SEQ ID NO:9 or SEQ ID NO:10, or the complement thereof; and/or iii) the nucleotide sequence of the SLC39A5 cDNA molecule produced from the mRNA in the biological sample, wherein the sequenced portion comprises a position corresponding to position 371 according to SEQ ID NO:16 or SEQ ID NO:17, or the complement thereof.
  • the sequenced portion of the SLC39A5 nucleic acid molecule in the biological sample comprises: an adenine or guanine at a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3, an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:9 or SEQ ID NO:10, or an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:16 or SEQ ID NO:17, then the SLC39A5 nucleic acid molecule in the biological sample is an SLC39A5 predicted loss-of-function variant nucleic acid molecule.
  • the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of: i) the nucleotide sequence of the SLC39A5 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 6,352 according to SEQ ID NO:5, or the complement thereof; ii) the nucleotide sequence of the SLC39A5 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 1,194 according to SEQ ID NO:12, or the complement thereof; and/or iii) the nucleotide sequence of the SLC39A5 cDNA molecule produced from the mRNA in the biological sample, wherein the sequenced portion comprises a position corresponding to position 1,194 according to SEQ ID NO:19, or the complement thereof.
  • the sequenced portion of the SLC39A5 nucleic acid molecule in the biological sample comprises: a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5, a uracil at a position corresponding to position 1,194 according to SEQ ID NO:12, or a thymine at a position corresponding to position 1,194 according to SEQ ID NO:19, then the SLC39A5 nucleic acid molecule in the biological sample is an SLC39A5 predicted loss- of-function variant nucleic acid molecule.
  • the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of: i) the nucleotide sequence of the SLC39A5 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 5,624 according to SEQ ID NO:7, or the complement thereof; ii) the nucleotide sequence of the SLC39A5 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 1,161 according to SEQ ID NO:14, or the complement thereof; and/or iii) the nucleotide sequence of the SLC39A5 cDNA molecule produced from the mRNA in the biological sample, wherein the sequenced portion comprises a position corresponding to position 1,161 according to SEQ ID NO:21, or the complement thereof.
  • the sequenced portion of the SLC39A5 nucleic acid molecule in the biological sample comprises: a thymine at a position corresponding to position 5,624 according to SEQ ID NO:7, a uracil at a position corresponding to position 1,161 according to SEQ ID NO:14, or a thymine at a position corresponding to position 1,161 according to SEQ ID NO:21, then the SLC39A5 nucleic acid molecule in the biological sample is an SLC39A5 predicted loss- of-function variant nucleic acid molecule.
  • the determining step, detecting step, or genotyping assay comprises: a) contacting the biological sample with a primer hybridizing to a portion of the nucleotide sequence of the SLC39A5: i) genomic nucleic acid molecule that is proximate to a position corresponding to position 5,604 according to SEQ ID NO:4; ii) mRNA molecule that is proximate to a position corresponding to position 1,141 according to SEQ ID NO:ll; and/or iii) cDNA molecule that is proximate to a position corresponding to position 1,141 according to SEQ ID NO:18; b) extending the primer at least through the position of the nucleotide sequence of the SLC39A5: i) genomic nucleic acid molecule corresponding to position 5,604 according to SEQ ID NO:4; ii) mRNA molecule corresponding to position 1,141 according to SEQ ID NO:ll; and/or iii) cDNA molecule
  • the determining step, detecting step, or genotyping assay comprises: a) contacting the biological sample with a primer hybridizing to a portion of the nucleotide sequence of the SLC39A5: i) genomic nucleic acid molecule that is proximate to a position corresponding to position 5,604 according to SEQ ID NO:6; ii) mRNA molecule that is proximate to a position corresponding to position 1,468 according to SEQ ID NO:13; and/or iii) cDNA molecule that is proximate to a position corresponding to position 1,468 according to SEQ ID NO:20; b) extending the primer at least through the position of the nucleotide sequence of the SLC39A5: i) genomic nucleic acid molecule corresponding to position 6,899 according to SEQ ID NO:6; ii) mRNA molecule corresponding to position 1,468 according to SEQ ID NO:13; and/or iii) cDNA
  • the determining step, detecting step, or genotyping assay comprises: a) contacting the biological sample with a primer hybridizing to a portion of the nucleotide sequence of the SLC39A5: i) genomic nucleic acid molecule that is proximate to a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3; ii) mRNA molecule that is proximate to a position corresponding to position 371 according to SEQ ID NO:9 or SEQ ID NO:10; and/or iii) cDNA molecule that is proximate to a position corresponding to position 371 according to SEQ ID NO:16 or SEQ ID NO:17; b) extending the primer at least through the position of the nucleotide sequence of the SLC39A5: i) genomic nucleic acid molecule corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3; ii) mRNA molecule
  • the determining step, detecting step, or genotyping assay comprises: a) contacting the biological sample with a primer hybridizing to a portion of the nucleotide sequence of the SLC39A5: i) genomic nucleic acid molecule that is proximate to a position corresponding to position 6,352 according to SEQ ID NO:5; ii) mRNA molecule that is proximate to a position corresponding to position 1,194 according to SEQ ID NO:12; and/or iii) cDNA molecule that is proximate to a position corresponding to position 1,194 according to SEQ ID NO:19; b) extending the primer at least through the position of the nucleotide sequence of the SLC39A5: i) genomic nucleic acid molecule corresponding to position 6,352 according to SEQ ID NO:5; ii) mRNA molecule corresponding to position 1,194 according to SEQ ID NO:12; and/or iii) cDNA
  • the determining step, detecting step, or genotyping assay comprises: a) contacting the biological sample with a primer hybridizing to a portion of the nucleotide sequence of the SLC39A5: i) genomic nucleic acid molecule that is proximate to a position corresponding to position 5,624 according to SEQ ID NO:7; ii) mRNA molecule that is proximate to a position corresponding to position 1,161 according to SEQ ID NO:14; and/or iii) cDNA molecule that is proximate to a position corresponding to position 1,161 according to SEQ ID NO:21; b) extending the primer at least through the position of the nucleotide sequence of the SLC39A5: i) genomic nucleic acid molecule corresponding to position 5,624 according to SEQ ID NO:7; ii) mRNA molecule corresponding to position 1,161 according to SEQ ID NO:14; and/or iii) cDNA
  • the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only an SLC39A5 genomic nucleic acid molecule is analyzed.
  • only an SLC39A5 mRNA is analyzed. In some embodiments, only an SLC39A5 cDNA obtained from SLC39A5 mRNA is analyzed.
  • the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of the nucleic acid molecule that encodes the human SLC39A5 polypeptide, wherein the amplified portion comprises: i) a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, or the complement thereof; ii) a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll, or the complement thereof; and/or iii) a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:18, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleic acid sequence of the amplified
  • the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of the nucleic acid molecule that encodes the human SLC39A5 polypeptide, wherein the amplified portion comprises: i) a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6, or the complement thereof; ii) a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:13, or the complement thereof; and/or iii) a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:20, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleic acid sequence of the amp
  • the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of the nucleic acid molecule that encodes the human SLC39A5 polypeptide, wherein the amplified portion comprises: i) an adenine or guanine at a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3, or the complement thereof; ii) an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:9 or SEQ ID NO:10, or the complement thereof; and/or iii) an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:16 or SEQ ID NO:17, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alter
  • the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of the nucleic acid molecule that encodes the human SLC39A5 polypeptide, wherein the amplified portion comprises: i) a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5, or the complement thereof; ii) a uracil at a position corresponding to position 1,194 according to SEQ ID NO:12, or the complement thereof; and/or iii) a thymine at a position corresponding to position 1,194 according to SEQ ID NO:19, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleic acid sequence of the amp
  • the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of the nucleic acid molecule that encodes the human SLC39A5 polypeptide, wherein the amplified portion comprises: i) a thymine at a position corresponding to position 5,624 according to SEQ ID NO:7, or the complement thereof; ii) a uracil at a position corresponding to position 1,161 according to SEQ ID NO:14, or the complement thereof; and/or iii) a thymine at a position corresponding to position 1,161 according to SEQ ID NO:21, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleic acid sequence of the
  • the nucleic acid molecule is mRNA and the determining step further comprises reverse-transcribing the mRNA into a cDNA prior to the amplifying step.
  • the determining step, detecting step, or genotyping assay comprises: contacting the nucleic acid molecule in the biological sample with an alteration- specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising: i) a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, or the complement thereof; ii) a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll, or the complement thereof; and/or a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:18, or the complement thereof; and detecting the detectable label.
  • the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising: i) a cytos
  • the determining step, detecting step, or genotyping assay comprises: contacting the nucleic acid molecule in the biological sample with an alteration- specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising: i) a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6, or the complement thereof; ii) a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:13, or the complement thereof; and/or a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:20, or the complement thereof; and detecting the detectable label.
  • the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising: i) a
  • the determining step, detecting step, or genotyping assay comprises: contacting the nucleic acid molecule in the biological sample with an alteration- specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising: i) an adenine or guanine at a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3, or the complement thereof; ii) an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:9 or SEQ ID NO:10, or the complement thereof; and/or an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:16 or SEQ ID NO:17, or the complement thereof; and detecting the detectable label.
  • the alteration-specific probe comprises a nucleotide sequence which hybridizes under
  • the determining step, detecting step, or genotyping assay comprises: contacting the nucleic acid molecule in the biological sample with an alteration- specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising: i) a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5, or the complement thereof; ii) a uracil at a position corresponding to position 1,194 according to SEQ ID NO:12, or the complement thereof; and/or a thymine at a position corresponding to position 1,194 according to SEQ ID NO:19, or the complement thereof; and detecting the detectable label.
  • the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising: i) a
  • the determining step, detecting step, or genotyping assay comprises: contacting the nucleic acid molecule in the biological sample with an alteration- specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising: i) a thymine at a position corresponding to position 5,624 according to SEQ ID NO:7, or the complement thereof; ii) a uracil at a position corresponding to position 1,161 according to SEQ ID NO:14, or the complement thereof; and/or a thymine at a position corresponding to position 1,161 according to SEQ ID NO:21, or the complement thereof; and detecting the detectable label.
  • the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising: i) a
  • Alteration-specific polymerase chain reaction techniques can be used to detect mutations such as SN Ps in a nucleic acid sequence. Alteration-specific primers can be used because the DNA polymerase will not extend when a mismatch with the template is present.
  • the nucleic acid molecule in the sample is rriRNA and the mRNA is reverse-transcribed into a cDNA prior to the amplifying step. In some embodiments, the nucleic acid molecule is present within a cell obtained from the human subject.
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule can be any SLC39A5 nucleic acid molecule (such as, for example, genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding an SLC39A5 polypeptide having a partial loss-of- function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function.
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule can be any nucleic acid molecule encoding SLC39A5 M304T, G413A, Y47Stop, R322Stop, or R311Stop.
  • the SLC39A5 predicted loss-of-function variant nucleic acid molecule encodes SLC39A5 Y47Stop, R322Stop, or R311Stop.
  • the assay comprises contacting the biological sample with a primer or probe, such as an alteration-specific primer or alteration-specific probe, that specifically hybridizes to an SLC39A5 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence and not the corresponding SLC39A5 reference sequence under stringent conditions, and determining whether hybridization has occurred.
  • a primer or probe such as an alteration-specific primer or alteration-specific probe
  • the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assays also comprise reverse transcribing mRNA into cDNA, such as by the reverse transcriptase polymerase chain reaction (RT-PCR).
  • RNA sequencing RNA-Seq
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the methods utilize probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and/or identify a polynucleotide comprising an SLC39A5 variant genomic nucleic acid molecule, variant mRNA molecule, or variant cDNA molecule.
  • the hybridization conditions or reaction conditions can be determined by the operator to achieve this result.
  • the nucleotide length may be any length that is sufficient for use in a detection method of choice, including any assay described or exemplified herein.
  • Such probes and primers can hybridize specifically to a target nucleotide sequence under high stringency hybridization conditions.
  • Probes and primers may have complete nucleotide sequence identity of contiguous nucleotides within the target nucleotide sequence, although probes differing from the target nucleotide sequence and that retain the ability to specifically detect and/or identify a target nucleotide sequence may be designed by conventional methods. Probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity with the nucleotide sequence of the target nucleic acid molecule.
  • an SLC39A5 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or complement thereof, within a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4 (genomic nucleic acid molecule), or a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll (mRNA molecule), or a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:18 (cDNA molecule)
  • the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from the 5' flanking sequence adjacent to a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, or a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll, or a cytos
  • the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. This distance can range from one nucleotide base pair up to the limits of the amplification reaction, or about twenty thousand nucleotide base pairs.
  • the primer pair flanks a region including positions comprising a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, or a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll, or a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:18, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of positions comprising a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, or a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll, or a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:18.
  • an SLC39A5 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or complement thereof, within a biological sample comprises a nucleotide sequence comprising a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6 (genomic nucleic acid molecule), or a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:13 (mRNA molecule), or a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:20 (cDNA molecule)
  • the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from the 5' flanking sequence adjacent to a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6, or a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:13, or a
  • the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. This distance can range from one nucleotide base pair up to the limits of the amplification reaction, or about twenty thousand nucleotide base pairs.
  • the primer pair flanks a region including positions comprising a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6, or a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:13, or a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:20, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of positions comprising a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6, or a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:13, or a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:20.
  • an SLC39A5 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule produced from an mRNA molecule), or complement thereof, within a biological sample comprises a nucleotide sequence comprising an adenine or guanine at a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3 (genomic nucleic acid molecule), or an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:9 or SEQ ID NO:10 (mRNA molecule), or an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:16 or SEQ ID NO:17 (cDNA molecule)
  • the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from the 5' flanking sequence adjacent to an adenine or guanine at a position corresponding
  • the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. This distance can range from one nucleotide base pair up to the limits of the amplification reaction, or about twenty thousand nucleotide base pairs.
  • the primer pair flanks a region including positions comprising an adenine or guanine at a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3, or an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:9 or SEQ ID NO:10, or an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:16 or SEQ ID NO:17, and at least 1, 2, 3, 4, 5, 6, 1, 8, 9, 10, or more nucleotides on each side of positions comprising an adenine or guanine at a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3, or an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:9 or SEQ ID NO:10, or an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:16 or
  • an SLC39A5 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or complement thereof, within a biological sample comprises a nucleotide sequence comprising a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5 (genomic nucleic acid molecule), or a uracil at a position corresponding to position 1,194 according to SEQ ID NO:12 (mRNA molecule), or a thymine at a position corresponding to position 1,194 according to SEQ ID NO:19 (cDNA molecule)
  • the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from the 5' flanking sequence adjacent to a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5, or a uracil at a position corresponding to position 1,194 according to SEQ ID NO:12, or a
  • the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. This distance can range from one nucleotide base pair up to the limits of the amplification reaction, or about twenty thousand nucleotide base pairs.
  • the primer pair flanks a region including positions comprising a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5, or a uracil at a position corresponding to position 1,194 according to SEQ ID NO:12, or a thymine at a position corresponding to position 1,194 according to SEQ ID NO:19, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of positions comprising a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5, or a uracil at a position corresponding to position 1,194 according to SEQ ID NO:12, or a thymine at a position corresponding to position 1,194 according to SEQ ID NO:19.
  • an SLC39A5 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or complement thereof, within a biological sample comprises a nucleotide sequence comprising a thymine at a position corresponding to position 5,624 according to SEQ ID NO:7 (genomic nucleic acid molecule), or a uracil at a position corresponding to position 1,161 according to SEQ ID NO:14 (mRNA molecule), or a thymine at a position corresponding to position 1,161 according to SEQ ID NO:21 (cDNA molecule)
  • the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from the 5' flanking sequence adjacent to a thymine at a position corresponding to position 5,624 according to SEQ ID NO:7, or a uracil at a position corresponding to position 1,161 according to SEQ ID NO:14, or
  • the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. This distance can range from one nucleotide base pair up to the limits of the amplification reaction, or about twenty thousand nucleotide base pairs.
  • the primer pair flanks a region including positions comprising a thymine at a position corresponding to position 5,624 according to SEQ ID NO:7, or a uracil at a position corresponding to position 1,161 according to SEQ ID NO:14, or a thymine at a position corresponding to position 1,161 according to SEQ ID NO:21, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of positions comprising a thymine at a position corresponding to position 5,624 according to SEQ ID NO:7, or a uracil at a position corresponding to position 1,161 according to SEQ ID NO:14, or a thymine at a position corresponding to position 1,161 according to SEQ ID NO:21.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose, such as the PCR primer analysis tool in Vector NTI version 10 (Informax Inc., Bethesda Md.); PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer3 (Version 0.4.0.COPYRGT., 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, the sequence can be visually scanned and primers manually identified using known guidelines.
  • nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing.
  • Other methods involve nucleic acid hybridization methods other than sequencing, including using labeled primers or probes directed against purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)).
  • FISH fluorescence in situ hybridization
  • a target nucleic acid molecule may be amplified prior to or simultaneous with detection.
  • nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA).
  • Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).
  • stringent conditions can be employed such that a probe or primer will specifically hybridize to its target.
  • a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence to a detectably greater degree than to other non-target sequences, such as, at least 2-fold, at least 3-fold, at least 4- fold, or more over background, including over 10-fold over background.
  • Stringent conditions are sequence-dependent and will be different in different circumstances.
  • stringent conditions for hybridization and detection will be those in which the salt concentration is less than about 1.5 M Na + ion, typically about 0.01 to 1.0 M Na + ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (such as, for example, 10 to 50 nucleotides) and at least about 60°C for longer probes (such as, for example, greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
  • the present disclosure also provides molecular complexes comprising any of the SLC39A5 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules), or complement thereof, described herein and any of the allele-specific primers or allele-specific probes described herein.
  • the SLC39A5 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules), or complement thereof, in the molecular complexes are single-stranded.
  • the SLC39A5 nucleic acid molecule is any of the genomic nucleic acid molecules described herein.
  • the SLC39A5 nucleic acid molecule is any of the mRNA molecules described herein.
  • the SLC39A5 nucleic acid molecule is any of the cDNA molecules described herein.
  • the molecular complex comprises any of the SLC39A5 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules), or complement thereof, described herein and any of the allele-specific primers described herein.
  • the molecular complex comprises any of the SLC39A5 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules), or complement thereof, described herein and any of the allele-specific probes described herein.
  • the complexes further comprise a non-human polymerase.
  • the molecular complex comprises an SLC39A5 variant genomic nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO:4, and the allele-specific primer or allele-specific probe that hybridizes to the cytosine at position 5,604 thereof.
  • the molecular complex comprises an SLC39A5 variant mRNA molecule comprising a nucleotide sequence according to SEQ ID NO:ll, and the allele-specific primer or allele-specific probe that hybridizes to the cytosine at position 1,141 thereof.
  • the molecular complex comprises an SLC39A5 variant cDNA molecule comprising a nucleotide sequence according to SEQ ID NO:18, and the allele-specific primer or allele- specific probe that hybridizes to the cytosine at position 1,141 thereof. ln some embodiments, the molecular complex comprises an SLC39A5 variant genomic nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO:6, and the allele-specific primer or allele-specific probe that hybridizes to the cytosine at position 6,899 thereof.
  • the molecular complex comprises an SLC39A5 variant mRNA molecule comprising a nucleotide sequence according to SEQ ID NO:13, and the allele-specific primer or allele-specific probe that hybridizes to the cytosine at position 1,468 thereof.
  • the molecular complex comprises an SLC39A5 variant cDNA molecule comprising a nucleotide sequence according to SEQ ID NO:20, and the allele-specific primer or allele- specific probe that hybridizes to the cytosine at position 1,468 thereof.
  • the molecular complex comprises an SLC39A5 variant genomic nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO:2 or SEQ ID NO:3, and the allele-specific primer or allele-specific probe that hybridizes to the adenine or guanine at position 1,353 thereof, respectively.
  • the molecular complex comprises an SLC39A5 variant mRNA molecule comprising a nucleotide sequence according to SEQ ID NO:9 or SEQ ID NO:10, and the allele-specific primer or allele-specific probe that hybridizes to the adenine or guanine at position 371 thereof, respectively.
  • the molecular complex comprises an SLC39A5 variant cDNA molecule comprising a nucleotide sequence according to SEQ ID NO:16 or SEQ ID NO:17, and the allele-specific primer or allele-specific probe that hybridizes to the adenine or guanine at position 371 thereof, respectively.
  • the molecular complex comprises an SLC39A5 variant genomic nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO:5, and the allele-specific primer or allele-specific probe that hybridizes to the thymine at position 6,352 thereof.
  • the molecular complex comprises an SLC39A5 variant mRNA molecule comprising a nucleotide sequence according to SEQ ID NO:12, and the allele-specific primer or allele-specific probe that hybridizes to the uracil at position 1,194 thereof.
  • the molecular complex comprises an SLC39A5 variant cDNA molecule comprising a nucleotide sequence according to SEQ ID NO:19, and the allele-specific primer or allele- specific probe that hybridizes to the thymine at position 1,194 thereof.
  • the molecular complex comprises an SLC39A5 variant genomic nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO:7, and the allele-specific primer or allele-specific probe that hybridizes to the thymine at position 5,624 thereof.
  • the molecular complex comprises an SLC39A5 variant mRNA molecule comprising a nucleotide sequence according to SEQ ID NO:14, and the allele-specific primer or allele-specific probe that hybridizes to the uracil at position 1,161 thereof.
  • the molecular complex comprises an SLC39A5 variant cDNA molecule comprising a nucleotide sequence according to SEQ ID NO:21, and the allele-specific primer or allele- specific probe that hybridizes to the thymine at position 1,161 thereof.
  • the present disclosure also provides methods of detecting the presence of a human SLC39A5 predicted loss-of-function polypeptide comprising performing an assay on a sample obtained from a human subject to determine whether an SLC39A5 polypeptide in the subject contains one or more variations that causes the polypeptide to have a loss-of-function (partial or complete) or predicted loss-of-function (partial or complete).
  • the methods detect the presence of SLC39A5 M304T, G413A, Y47Stop, R322Stop, or R311Stop.
  • the methods detect the presence of SLC39A5 Y47Stop, R322Stop, or R311Stop.
  • the methods comprise performing an assay on a sample obtained from a human subject to determine whether an SLC39A5 polypeptide in the sample comprises a threonine at a position corresponding to position 304 according to SEQ ID NO:24.
  • the methods comprise performing an assay on a sample obtained from a human subject to determine whether an SLC39A5 polypeptide in the sample comprises a alanine at a position corresponding to position 413 according to SEQ ID NO:26.
  • the methods comprise performing an assay on a sample obtained from a human subject to determine whether the sample contains an SLC39A5 truncated variant polypeptide terminating at a position corresponding to position 46 according to SEQ ID NO:23.
  • the SLC39A5 truncated variant polypeptide lacks amino acids at positions corresponding to positions 47 to 540 according to SEQ ID NO:22.
  • the SLC39A5 truncated variant polypeptide comprises or consists of SEQ ID NO:23.
  • the methods comprise performing an assay on a sample obtained from a human subject to determine whether the sample contains an SLC39A5 truncated variant polypeptide terminating at a position corresponding to position 321 according to SEQ ID NO:25.
  • the SLC39A5 truncated variant polypeptide lacks amino acids at positions corresponding to positions 322 to 540 according to SEQ ID NO:22.
  • the SLC39A5 truncated variant polypeptide comprises or consists of SEQ ID NO:25.
  • the methods comprise performing an assay on a sample obtained from a human subject to determine whether the sample contains an SLC39A5 truncated variant polypeptide terminating at a position corresponding to position 310 according to SEQ ID NO:27.
  • the SLC39A5 truncated variant polypeptide lacks amino acids at positions corresponding to positions 311 to 540 according to SEQ ID NO:22.
  • the SLC39A5 truncated variant polypeptide comprises or consists of SEQ ID NO:27.
  • the detecting step comprises sequencing at least a portion of the polypeptide that comprises a position corresponding to position 304 according to SEQ ID NO:22 or SEQ ID NO:24. In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide that comprises a position corresponding to position 413 according to SEQ ID NO:22 or SEQ ID NO:26. In some embodiments, the detecting step comprises sequencing the entire polypeptide.
  • the sequenced SLC39A5 polypeptide comprises or consists of SEQ ID NO:23. In some embodiments, the sequenced SLC39A5 polypeptide consists of SEQ ID NO:23. In some embodiments, the detecting step comprises sequencing at least a portion of an SLC39A5 polypeptide that may comprise positions corresponding to any positions that are C- terminal to 46 according to SEQ ID NO:23. If amino acids are detected in the SLC39A5 polypeptide at positions corresponding to positions 47 to 540 according to SEQ ID NO:22, then such SLC39A5 polypeptide is an SLC39A5 reference polypeptide.
  • the detecting step comprises sequencing the entire polypeptide.
  • the sequenced SLC39A5 polypeptide comprises or consists of SEQ ID NO:25. In some embodiments, the sequenced SLC39A5 polypeptide consists of SEQ ID NO:25. In some embodiments, the detecting step comprises sequencing at least a portion of an SLC39A5 polypeptide that may comprise positions corresponding to any positions that are C- terminal to 321 according to SEQ ID NO:25. If amino acids are detected in the SLC39A5 polypeptide at positions corresponding to positions 322 to 540 according to SEQ ID NO:22, then such SLC39A5 polypeptide is an SLC39A5 reference polypeptide.
  • the detecting step comprises sequencing the entire polypeptide.
  • the sequenced SLC39A5 polypeptide comprises or consists of SEQ ID NO:27. In some embodiments, the sequenced SLC39A5 polypeptide consists of SEQ ID NO:27. In some embodiments, the detecting step comprises sequencing at least a portion of an SLC39A5 polypeptide that may comprise positions corresponding to any positions that are C- terminal to 310 according to SEQ ID NO:27. If amino acids are detected in the SLC39A5 polypeptide at positions corresponding to positions 311 to 540 according to SEQ ID NO:22, then such SLC39A5 polypeptide is an SLC39A5 reference polypeptide.
  • the detecting step comprises sequencing the entire polypeptide.
  • the detecting step comprises an immunoassay for detecting the presence of a polypeptide that comprises a position corresponding to position 304 according to SEQ ID NO:22 or SEQ ID NO:24. In some embodiments, the detecting step comprises an immunoassay for detecting the presence of a polypeptide that comprises a position corresponding to position 413 according to SEQ ID NO:22 or SEQ ID NO:26.
  • the detecting step comprises an immunoassay for detecting the presence of an SLC39A5 polypeptide that comprises or consists of SEQ ID NO:23.
  • the SLC39A5 polypeptide consists of SEQ ID NO:23.
  • the detecting step comprises detecting at least a portion of an SLC39A5 polypeptide that may comprise positions corresponding to any positions that are C-terminal to 46 according to SEQ ID NO:23. If amino acids are detected in the SLC39A5 polypeptide at positions corresponding to positions 47 to 540 according to SEQ ID NO:22, then such SLC39A5 polypeptide is an SLC39A5 reference polypeptide.
  • the detecting step comprises an immunoassay for detecting the presence of an SLC39A5 polypeptide that comprises or consists of SEQ ID NO:25.
  • the SLC39A5 polypeptide consists of SEQ ID NO:25.
  • the detecting step comprises detecting at least a portion of an SLC39A5 polypeptide that may comprise positions corresponding to any positions that are C-terminal to 321 according to SEQ ID NO:25. If amino acids are detected in the SLC39A5 polypeptide at positions corresponding to positions 322 to 540 according to SEQ ID NO:22, then such SLC39A5 polypeptide is an SLC39A5 reference polypeptide. A lack of detection of positions 322 to 540 according to SEQ ID NO:22 in the SLC39A5 polypeptide indicates that the SLC39A5 polypeptide terminates at position 321 according to SEQ ID NO:25 and is an SLC39A5 predicted loss-of-function polypeptide.
  • the detecting step comprises an immunoassay for detecting the presence of an SLC39A5 polypeptide that comprises or consists of SEQ ID NO:27.
  • the SLC39A5 polypeptide consists of SEQ ID NO:27.
  • the detecting step comprises detecting at least a portion of an SLC39A5 polypeptide that may comprise positions corresponding to any positions that are C-terminal to 310 according to SEQ ID NO:27. If amino acids are detected in the SLC39A5 polypeptide at positions corresponding to positions 311 to 540 according to SEQ ID NO:22, then such SLC39A5 polypeptide is an SLC39A5 reference polypeptide.
  • a lack of detection of positions 311 to 540 according to SEQ ID NO:22 in the SLC39A5 polypeptide indicates that the SLC39A5 polypeptide terminates at position 310 according to SEQ ID NO:27 and is an SLC39A5 predicted loss-of-function polypeptide.
  • the human subject when the human subject does not have an SLC39A5 predicted loss-of-function polypeptide, then the human subject has an increased risk for developing increased serum glucose level and/or hyperglycemia; and when the human subject has an SLC39A5 predicted loss-of-function polypeptide, then the human subject has a decreased risk for developing increased serum glucose level and/or hyperglycemia.
  • the probes and/or primers (including alteration-specific probes and alteration-specific primers) described herein comprise or consist of from about 15 to about 100, from about 15 to about 35 nucleotides.
  • the alteration-specific probes and alteration-specific primers comprise DNA.
  • the alteration-specific probes and alteration-specific primers comprise RNA.
  • the probes and primers described herein (including alteration-specific probes and alteration-specific primers) have a nucleotide sequence that specifically hybridizes to any of the nucleic acid molecules disclosed herein, or the complement thereof.
  • the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.
  • specifically hybridizes means that the probe or primer (including alteration-specific probes and alteration-specific primers) does not hybridize to a nucleic acid sequence encoding an SLC39A5 reference genomic nucleic acid molecule, an SLC39A5 reference mRNA molecule, and/or an SLC39A5 reference cDNA molecule.
  • the probes (such as, for example, an alteration-specific probe) comprise a label.
  • the label is a fluorescent label, a radiolabel, or biotin.
  • SLC39A5 reference genomic nucleic acid molecule The nucleotide sequence of an SLC39A5 reference genomic nucleic acid molecule is set forth in SEQ ID NO:l.
  • position 5,604 is a thymine.
  • position 6,899 is a guanine.
  • position 1,353 is a cytosine
  • codon positions 1,351 to 1,353 is a TAC codon.
  • position 6,352 is a cytosine
  • codon positions 6,352 to 6,354 is a CGA codon.
  • position 5,624 is a cytosine
  • codon positions 5,624 to 5,626 is a CGA codon.
  • a variant genomic nucleic acid molecule of SLC39A5 exists, wherein the thymine at position 5,604 is replaced with cytosine.
  • the nucleotide sequence of this SLC39A5 variant genomic nucleic acid molecule is set forth in SEQ ID NO:4.
  • Another variant genomic nucleic acid molecule of SLC39A5 exists, wherein the guanine at position 6,899 is replaced with cytosine.
  • the nucleotide sequence of this SLC39A5 variant genomic nucleic acid molecule is set forth in SEQ ID NO:6.
  • SLC39A5 Another variant genomic nucleic acid molecule of SLC39A5 exists, wherein the cytosine at position 1,353 according to SEQ ID NO:l is replaced with adenine or guanine, and the TAC codon at positions 1,351 to 1,353 according to SEQ ID NO:l is replaced with TAA or TAG.
  • the nucleotide sequence of this SLC39A5 variant genomic nucleic acid molecule is set forth in SEQ ID NO:2 or SEQ ID NO:3.
  • Another variant genomic nucleic acid molecule of SLC39A5 exists, wherein the cytosine at position 6,352 according to SEQ ID NO:l is replaced with thymine, and the CGA codon at positions 6,352 to 6,354 according to SEQ ID NO:l is replaced with TGA.
  • the nucleotide sequence of this SLC39A5 variant genomic nucleic acid molecule is set forth in SEQ ID NO:5.
  • Another variant genomic nucleic acid molecule of SLC39A5 exists, wherein the cytosine at position 5,624 according to SEQ ID NO:l is replaced with thymine, and the CGA codon at positions 5,624 to 5,626 according to SEQ ID NO:l is replaced with TGA.
  • the nucleotide sequence of this SLC39A5 variant genomic nucleic acid molecule is set forth in SEQ ID NO:7.
  • SEQ ID NO:8 The nucleotide sequence of an SLC39A5 reference mRNA molecule is set forth in SEQ ID NO:8.
  • position 1,141 is a uracil.
  • position 1,468 is a guanine.
  • position 371 is a cytosine, and codon positions 369 to 371 is a UAC codon.
  • position 1,194 is a cytosine, and codon positions 1,194 to 1,196 is a CGA codon.
  • position 1,161 is a cytosine
  • codon positions 1,161 to 1,163 is a CGA codon.
  • a variant mRNA molecule of SLC39A5 exists, wherein the uracil at position 1,141 is replaced with cytosine.
  • the nucleotide sequence of this SLC39A5 variant mRNA molecule is set forth in SEQ ID NO:ll.
  • Another variant mRNA molecule of SLC39A5 exists, wherein the guanine at position 1,468 is replaced with cytosine.
  • the nucleotide sequence of this SLC39A5 variant mRNA molecule is set forth in SEQ ID NO:13.
  • Another variant mRNA molecule of SLC39A5 exists, wherein the cytosine at position 371 according to SEQ ID NO:8 is replaced with adenine or guanine, and the UAC codon at positions 369 to 371 according to SEQ ID NO:8 is replaced with UAA or UAG.
  • the nucleotide sequence of this SLC39A5 variant mRNA molecule is set forth in SEQ ID NO:9 or SEQ ID NO:10.
  • Another variant mRNA molecule of SLC39A5 exists, wherein the cytosine at position 1,194 according to SEQ ID NO:8 is replaced with uracil, and the CGA codon at positions 1,194 to 1,196 according to SEQ ID NO:8 is replaced with UGA.
  • SLC39A5 variant mRNA molecule The nucleotide sequence of this SLC39A5 variant mRNA molecule is set forth in SEQ ID NO:12.
  • Another variant mRNA molecule of SLC39A5 exists, wherein the cytosine at position 1,161 according to SEQ ID NO:8 is replaced with uracil, and the CGA codon at positions 1,161 to 1,163 according to SEQ ID NO:8 is replaced with UGA.
  • SEQ ID NO:14 The nucleotide sequence of this SLC39A5 variant mRNA molecule is set forth in SEQ ID NO:14.
  • SEQ ID NO:15 The nucleotide sequence of an SLC39A5 reference cDNA molecule is set forth in SEQ ID NO:15.
  • position 1,141 is a thymine.
  • position 1,468 is a guanine.
  • position 371 is a cytosine, and codon positions 369 to 371 is a TAC codon.
  • position 1,194 is a cytosine, and codon positions 1,194 to 1,196 is a CGA codon.
  • position 1,161 is a cytosine
  • codon positions 1,161 to 1,163 is a CGA codon.
  • a variant cDNA molecule of SLC39A5 exists, wherein the thymine at position 1,141 is replaced with cytosine.
  • the nucleotide sequence of this SLC39A5 variant cDNA molecule is set forth in SEQ ID NO:18.
  • Another variant cDNA molecule of SLC39A5 exists, wherein the guanine at position 1,468 is replaced with cytosine.
  • the nucleotide sequence of this SLC39A5 variant cDNA molecule is set forth in SEQ ID NO:20.
  • Another variant cDNA molecule of SLC39A5 exists, wherein the cytosine at position 371 according to SEQ ID NO:15 is replaced with adenine or guanine, and the TAC codon at positions 369 to 371 according to SEQ ID NO:15 is replaced with TAA or TAG.
  • the nucleotide sequence of this SLC39A5 variant cDNA molecule is set forth in SEQ ID NO:16 or SEQ ID NO:17.
  • Another variant cDNA molecule of SLC39A5 exists, wherein the cytosine at position 1,194 according to SEQ ID NO:15 is replaced with thymine, and the CGA codon at positions 1,194 to 1,196 according to SEQ ID NO:15 is replaced with TGA.
  • SLC39A5 variant cDNA molecule The nucleotide sequence of this SLC39A5 variant cDNA molecule is set forth in SEQ ID NO:19.
  • Another variant cDNA molecule of SLC39A5 exists, wherein the cytosine at position 1,161 according to SEQ ID NO:15 is replaced with thymine, and the CGA codon at positions 1,161 to 1,163 according to SEQ ID NO:15 is replaced with TGA.
  • the nucleotide sequence of this SLC39A5 variant cDNA molecule is set forth in SEQ ID NO:21.
  • SLC39A5 reference polypeptide The amino acid sequence of an SLC39A5 reference polypeptide is set forth in SEQ ID NO:22.
  • the SLC39A5 reference polypeptide is 540 amino acids in length.
  • position 304 is methionine.
  • position 413 is glycine.
  • position 47 is tyrosine.
  • position 322 is an arginine.
  • position 311 is an arginine.
  • SLC39A5 variant polypeptide exists (M304T or Met304Thr), the amino acid sequence of which is set forth in SEQ ID NO:24. Referring to SEQ ID NO:24, the SLC39A5 variant polypeptide is 540 amino acids in length. Referring to SEQ ID NO:24, position 304 is threonine.
  • Another SLC39A5 variant polypeptide exists (G413A or Gly413Ala), the amino acid sequence of which is set forth in SEQ ID NO:26. Referring to SEQ ID NO:26, the SLC39A5 variant polypeptide is 540 amino acids in length. Referring to SEQ ID NO:26, position 413 is alanine.
  • SLC39A5 truncated variant polypeptide exists (Y47Stop or Tyr47Stop), the amino acid sequence of which is set forth in SEQ ID NO:23.
  • the SLC39A5 variant polypeptide is 46 amino acids in length.
  • the SLC39A5 variant polypeptide is truncated at position 46 and does not contain amino acids at positions corresponding to positions 47 to 540 according to SEQ ID NO:22.
  • Another SLC39A5 truncated variant polypeptide exists (R322Stop or Arg322Stop), the amino acid sequence of which is set forth in SEQ ID NO:25.
  • the SLC39A5 variant polypeptide is 321 amino acids in length. Referring to SEQ ID NO:25, the SLC39A5 variant polypeptide is truncated at position 321 and does not contain amino acids at positions corresponding to positions 322 to 540 according to SEQ ID NO:22. Another SLC39A5 truncated variant polypeptide exists (R311Stop or
  • SLC39A5 variant polypeptide is 310 amino acids in length.
  • SLC39A5 variant polypeptide is truncated at position 310 and does not contain amino acids at positions corresponding to positions 311 to 540 according to SEQ ID NO:22.
  • nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids.
  • the nucleotide sequences follow the standard convention of beginning at the 5' end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
  • the amino acid sequence follows the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.
  • the phrase "corresponding to" or grammatical variations thereof when used in the context of the numbering of a particular nucleotide or nucleotide sequence or position refers to the numbering of a specified reference sequence when the particular nucleotide or nucleotide sequence is compared to a reference sequence (such as, for example, SEQ ID NO:l, SEQ ID NO:8, or SEQ ID NO:15).
  • a reference sequence such as, for example, SEQ ID NO:l, SEQ ID NO:8, or SEQ ID NO:15.
  • the residue (such as, for example, nucleotide or amino acid) number or residue (such as, for example, nucleotide or amino acid) position of a particular polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the particular nucleotide or nucleotide sequence.
  • a particular nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences.
  • the gaps are present, the numbering of the residue in the particular nucleotide or nucleotide sequence is made with respect to the reference sequence to which it has been aligned.
  • a nucleic acid molecule comprising a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4 means that if the nucleotide sequence of the SLC39A5 genomic nucleic acid molecule is aligned to the sequence of SEQ ID NO:4, the SLC39A5 sequence has a cytosine residue at the position that corresponds to position 5,604 according to SEQ ID NO:4.
  • mRNA molecules comprising a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll
  • cDNA molecules comprising a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:18.
  • these phrases refer to a nucleic acid molecule encoding an SLC39A5 polypeptide, wherein the genomic nucleic acid molecule has a nucleotide sequence that comprises a cytosine residue that is homologous to the cytosine residue at position 5,604 according to SEQ ID NO:4 (or wherein the mRNA molecule has a nucleotide sequence that comprises a cytosine residue that is homologous to the cytosine residue at position 1,141 according to SEQ ID NO:ll, or wherein the cDNA molecule has a nucleotide sequence that comprises a cytosine residue that is homologous to the cytosine residue at position 1,141 according to SEQ ID NO:18).
  • such a sequence is also referred to as "SLC39A5 sequence with the M304T alteration” or “SLC39A5 sequence with the M304T variation” referring to genomic nucleic acid molecules (or “SLC39A5 sequence with the U1,141C alteration” or “SLC39A5 sequence with the U1,141C variation” referring to mRNA molecules, and “SLC39A5 sequence with the T1,141C alteration” or “SLC39A5 sequence with the T1,141C variation” referring to cDNA molecules).
  • a position within an SLC39A5 genomic nucleic acid molecule that corresponds to position 5,604 according to SEQ ID NO:4, for example, can be identified by performing a sequence alignment between the nucleotide sequence of a particular SLC39A5 nucleic acid molecule and the nucleotide sequence of SEQ ID NO:4.
  • sequence alignments may be performed. However, sequences can also be aligned manually.
  • the present disclosure also provides therapeutic agents that treat or inhibit increased serum glucose level and/or hyperglycemia for use in the treatment of increased serum glucose level and/or hyperglycemia (or for use in the preparation of a medicament for treating increased serum glucose level and/or hyperglycemia) in a human subject, wherein the human subject has: a genomic nucleic acid molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, or the complement thereof; an mRNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide,
  • the present disclosure also provides SLC39A5 inhibitors for use in the treatment of increased serum glucose level and/or hyperglycemia (or for use in the preparation of a medicament for treating increased serum glucose level and/or hyperglycemia) in a human subject, wherein the human subject has: a genomic nucleic acid molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 5,604 according to SEQ ID NO:4, or the complement thereof; an mRNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 1,141 according to SEQ ID NO:ll, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises
  • the human subject has: a genomic nucleic acid molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 6,899 according to SEQ ID NO:6, or the complement thereof; an mRNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:13, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 1,468 according to SEQ ID NO:20, or the complement thereof; or an SLC39A5 polypeptide that comprises a alanine at a position corresponding to position 413 according
  • the human subject has: a genomic nucleic acid molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises an adenine or guanine at a position corresponding to position 1,353 according to SEQ ID NO:2 or SEQ ID NO:3, or the complement thereof; an mRNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:9 or SEQ ID NO:10, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises an adenine or guanine at a position corresponding to position 371 according to SEQ ID NO:16 or SEQ ID NO:17, or the complement
  • the human subject has: a genomic nucleic acid molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 6,352 according to SEQ ID NO:5, or the complement thereof; an mRNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 1,194 according to SEQ ID NO:12, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,194 according to SEQ ID NO:19, or the complement thereof; or an SLC39A5 polypeptide that is truncated at position 321 according to SEQ ID NO:25
  • the human subject has: a genomic nucleic acid molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 5,624 according to SEQ ID NO:7, or the complement thereof; an mRNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 1,161 according to SEQ ID NO:14, or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a human SLC39A5 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 1,161 according to SEQ ID NO:21, or the complement thereof; or an SLC39A5 polypeptide that is truncated at position 310 according to SEQ ID NO:
  • the SLC39A5 gene was expressed using the IRES-dsRED reporter system transfected into cells, and the expression was verified. One day after transfection, the cells were shifted to Zn-free media, and a day later were perfused with buffer containing (0-1 mM) free Zn 2+ with or without TREN/Zn-Pyr (a cell permeable, high-affinity heavy metal (Zn 2+ >Fe 2+ >Mn 2+ ) chelator that exhibits low affinity for Mg 2+ and Ca 2+ ). The zinc-dependent transcriptional responses were monitored through expression of luciferase reporter gene driven by a metal responsive element (MRE). The results showed that SLC35 is required for Zn 2+ mediated MRE activation.
  • MRE metal responsive element
  • Example 3 Zinc Supplementation Rescues Hepatic Steatosis and Glucose Tolerance in DIO
  • Example 5 Loss of Slc39a5 results in elevated circulating zinc levels.
  • Lepr knockout had no effect on the increase of serum zinc resulting from SLC39A knockout ( Figures 7C and 7F).
  • Example 7 Slc39a5 LOF Rescues Hyperglycemia in Leptin Receptor Deficient (db/db, Female) Mice with Concomitant Increase in Ketogenesis
  • Example 8 SLC39A5 Heterozygous LOF have Elevated Serum Zinc
  • Slc39a5 loss of function mice The genetically engineered Slc39a5 /_ mouse strain was created using VelociGene ® technology. Briefly, C57BI/6NTac embryonic stem cells (ESC) were targeted for ablation of a portion of the Slc39a5 locus, beginning just after the start ATG and ending 5 base pairs before the 3' end of coding exon 2. This region contains the Slc39a5 signal peptide and much of the N- terminal extracellular domain. A lacZ reporter module was inserted in frame with the Slc39a5 start, followed by a fLoxed neomycin resistance cassette for selection in ESC. The resistance cassette was deleted prior to microinjection using self-deleting technology.
  • ESC C57BI/6NTac embryonic stem cells
  • the targeted cells were microinjected into 8-cell embryos from Charles River Laboratories Swiss Webster albino mice, yielding F0 VelociMice ® that were 100% derived from the targeted cells. These mice were subsequently bred to homozygosity and maintained in an animal facility during the whole period. Slc39a5 /+ heterozygous mice and C57BI/6NTac wildtype littermates were used as controls.
  • mice homozygous for Slc39a5 loss of function and wild-type littermates were co housed in a controlled environment (12 hour light/dark cycle, 22 ⁇ 1°C, 60-70% humidity) and fed ad-libitum with a high fat diet (Test Diet, Catalog # 9GWP) containing 35 ppm zinc starting at 6 weeks of age. Both male and female mice were used in this study. Upon an overnight fast (lasting 16 hours), serum and hepatic zinc along ALT and AST were assessed at 40 weeks of high fat diet challenge.
  • a separate cohort of age matched wild-type C57BLK/6 mice maintained on normal chow (Lab Diet, Catalog # 5K52) containing 85 ppm zinc were obtained from Jackson Laboratories as controls.
  • Serum and hepatic zinc analyses was conducted using flame atomic absorption spectroscopy as discussed below.
  • Serum ALT and AST levels and other liver and lipid traits were measured using ADVIA Chemistry XPT System (Siemens Healthineers).
  • ADVIA Chemistry XPT System Siemens Healthineers
  • explanted liver samples were fixed in 10% phosphate buffered formalin acetate at 4°C overnight, thoroughly rinsed in phosphate-buffered saline and embedded in paraffin wax.
  • Hematoxylin and eosin staining performed on 5 pm thick paraffin sections using standard histochemistry techniques. Sections were imaged using a 20X or 40X objective using the EVOS FL Auto microscope (Thermo Fisher Scientific).
  • Assays were performed on Siemens ADVIA Chemistry XPT.
  • the liver and lipid profile contained the following reagents: Alanine Aminotransferase (ALT, Siemens REF 03036926), Aspartate Aminotransferase (AST, Siemens REF 07499718), Cholesterol (CHOL, Siemens REF 10376501), Direct H DL Cholesterol (DH DL, Siemens REF 07511947), LDL Cholesterol Direct (DLDL, Siemens REF 09793248), Non-Esterified Fatty Acids (N EFA, Wako 999-34691, 995-34791, 991-34891, 993-35191), Triglycerides (TRIG, Siemens REF 10335892).
  • microcentrifuge tubes and all reagent mixing, assay timing, absorbance and concentration calculation was performed by the analyzer.
  • Figures 11, 12, and 13 depict serum zinc, liver zinc, alanine aminotransferase, and aspartate aminotransferase levels of Slc39a5 loss-of -function mice at 40 weeks on a high-fat diet regimen.
  • Female homozygous loss-of-function mice had elevated serum zinc and liver zinc levels as compared to wild-type mice on a normal chow (NC) or a high-fat diet (HFD). No differences were observed in male mice at this age.
  • female homozygous loss-of- function mice had reduced alanine aminotransferase levels as compared to wild-type mice on a normal chow (NC) or a high-fat diet (HFD).
  • mice had dramatic reduction in hepatic lipid accumulation as compared to wild-type mice on a normal chow (NC) or a high-fat diet (HFD) at 40 weeks based on hematoxylin and eosin staining and Masson's staining.
  • Male homozygous loss-of- function mice also showed modest reduction in hepatic lipid accumulation as compared to male wild-type littermates on a normal chow (NC) or on a high-fat diet (HFD) at 40 weeks based on hematoxylin and eosin staining and Masson's staining.
  • b-hydroxybutyrate assay buffer as per the manufacturer's recommendations. Homogenized samples were centrifuged at 13000 g for 10 minutes at 4°C, and the resulting supernatant was used for downstream analyses. In this assay, b-hydroxy butyrate concentration was determined by a coupled enzyme reaction which results in a colorimetric (450 nm) product proportional to the b-hydroxy butyrate present.
  • Assays were performed using Triglyceride Colorimetric Assay Kit (Cayman 10010303). Explanted liver samples (400 mg/sample) were homogenized in 2 ml of N P-40 Substitute Assay Reagent containing protease inhibitors. Homogenized samples were centrifuged at 10000 g for 10 minutes at 4°C, and the resulting supernatant was diluted at least 1:5 using N P-40 Substitute Assay Reagent for downstream analyses. This assay involves enzymatic hydrolysis of triglycerides by lipase to produce glycerol and free fatty acids. The glycerol released is subsequently measured by a coupled colorimetric enzymatic reaction. The absorbance was measured at 530-550 nm.
  • Figure 14 depicts hepatic triglyceride and b-hydroxybutyrate levels of Slc39a5 loss-of- function female mice at 40 weeks on a high-fat diet regimen.
  • Female homozygous loss-of- function mice had reduced hepatic triglyceride as compared to wild-type mice on a normal chow (NC) or a high-fat diet (H FD).
  • NC normal chow
  • H FD high-fat diet
  • female homozygous loss-of-function mice had elevated hepatic b-hydroxy butyrate levels as compared to wild-type mice on a normal chow (NC) or a high-fat diet (HFD).
  • mice Liver, lipid and glycemic traits of mice homozygous for Slc39a5 and leptin-receptor (Lepr) loss- of-function:
  • mice homozygous for Slc39a5 and Lepr loss of function and littermate controls were co-housed in a controlled environment (12 hour light/dark cycle, 22 ⁇ 1°C, 60-70% humidity) and fed ad-libitum with normal chow (PicoLab Rodent Diet 20, Catalog # 5053) containing 87 ppm zinc. Mice were monitored for health and growth kinetics periodically. Upon an overnight fast (lasting 16 hours), serum ALT and AST along with DLDL and fasting blood glucose were measured when the mice were 22 weeks of age.
  • Blood glucose was evaluated using AlphaTrak blood glucose monitoring system (Zoetis United States, Parsippany NJ) by sampling blood from the lateral tail vein. Liver and lipid traits were measured using a Siemens ADVIA Chemistry XPT as described previously. An oral glucose tolerance test was administered upon an overnight fast (lasting 16 hours) at 20 weeks of age by administering 2 g/kg of body weight of Dextrose (Hospira lnc 7 N DC 0409-4902-34) by oral gavage. Blood glucose was evaluated at defined time points (0, 15, 30, 60, and 120 minutes) using AlphaTrak blood glucose monitoring system (Zoetis United States, Parsippany NJ) by sampling blood from the lateral tail vein.
  • AlphaTrak blood glucose monitoring system Zoetis United States, Parsippany NJ
  • mice homozygous for Slc39a5 loss of function were bred in to a leptin-receptor deficient (Lepr /_ ) background (a commonly used rodent model of obesity induced
  • Leptin-receptor deficient mice were hyperglycemic and displayed elevated serum low-density lipoprotein levels. Furthermore, these mice had elevated serum ALT and AST levels suggesting impaired liver function at 22 weeks of age. Loss of Slc39a5 function in female Lepr receptor deficient mice significantly reduced fasting blood glucose, serum LDL-C, serum ALT and AST levels potentially ameliorating the obesity induced metabolic dysregulation in leptin-receptor deficient (see, Figures 15 and 16). In line with these
  • mice homozygous for Slc39a5 and Lepr loss of function demonstrated improved glucose tolerance when challenged with an oral glucose load.
  • Liver protein was extracted using RIPA buffer (Cell signaling technology, Cat#9806) with Halt Protease & Phosphatase Inhibitor Cocktail (ThermoFisher Scientific, Cat#78440). Five microgram proteins of each sample were separated in NuPAGE 4-12% Bis-Tris protein gel (Invitrogen, Cat#WG1403BOX), and transferred to nitrocellulose membrane using Trans-Blot ® TurboTM Transfer System (BioRad).
  • Blotting was performed using the following Cell Signaling Technology antibodies: Phospho-AM RKa (Thrl72) (Cell signaling technology, Cat#2535), AMPKa (Cell signaling technology, Cat#5831), Phospho-AMRKbI (Serl82) (Cell signaling technology, Cat#4186), SCD1 (Cell signaling technology, Cat#2794), Phospho-Acetyl CoA Carboxylase (Ser79) (Millipore Sigma, Cat#07-303), Acetyl CoA Carboxylase 1 (Millipore Sigma, Cat#MABS830), b- Actin (Sigma, Cat#5441), rabbit IgG conjugated to horseradish peroxidase (H RP) (Cell signaling technology, Cat#7074) and mouse IgG conjugated to H RP (Cell signaling technology, Cat#7076). Blots were developed using SuperSignal West Femto Substrate (ThermoFisher Scientific, Cat# 340
  • mice had elevated hepatic AMPK activation as observed by increased rAMRKa/AM RKa levels as compared to wild-type mice on a normal chow (NC) or on a high-fat diet (H FD).
  • increased AMPK activation in these mice resulted in increased phosphorylation of acetyl-coA carboxylase (observed by increased pACC/ACC levels) as compared to wild-type mice on a normal chow (NC) or on a high- fat diet (H FD).
  • SCD1 stearoyl-CoA desaturase
  • the immunogenic peptide fragment of SLC39A5 for mouse inoculation was selected based on the hydrophilicity of the amino acid sequence (see, Figure 18).
  • a mouse was inoculated with the immunogenic SLC39A5 fragment.
  • the mouse was sacrificed, and the splenocytes of the mouse were isolated.
  • the splenocytes were fused with hybridoma partner cells.
  • the successfully fused cells were diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture was continued.
  • Antibody-producing clones were identified by assaying the reactivity of antibody in the supernatant fluid of the wells with H EK cells overexpressing mouse SLC39A5 (see, Figure 19) and H EK cells overexpressing human SLC39A5 (see, Figure 20). Three positive clones that were reactive against both human and mouse SLC39A5 (see, 10D1, 10D4, a nd 11C1) were expanded and their monoclonal antibody product was selected.
  • mice homozygous or heterozygous for Slc39a5 loss of function and wild-type littermates were co-housed in a controlled environment (12 hour light/dark cycle, 22 ⁇ 1°C, 60- 70% humidity) and fed ad-libitum with a high fat high fructose diet (Test Diet, Catalog # 5WK9) or a control diet (Test Diet, Catalog# 58Y2) containing 35 ppm zinc starting at 10 weeks of age. Both male and female mice were used in this study. Longitudinal assessment of serum zinc, fasting blood glucose along with alanine aminotransferase (ALT) and aspartate
  • AST aminotransferase
  • mice homozygous for Slc39a5 and Lepr loss of function Lepr ⁇ ) and littermate controls (wildtype, 5/c39a5 / , Lepr ⁇ ) were co-housed in a controlled environment (12 hour light/dark cycle, 22 ⁇ 1°C, 60-70% humidity) and fed ad-libitum with normal chow (PicoLab Rodent Diet 20, Catalog # 5053) containing 87 ppm zinc. Mice were monitored for health and growth kinetics periodically. Upon an overnight fast (lasting 16 hours), serum ALT and AST (biomarkers of liver injury) along with DLDL and fasting blood glucose were measured when the mice were 22 weeks of age.
  • Blood glucose was evaluated using AlphaTrak blood glucose monitoring system (Zoetis U nited States, Parsippany NJ) by sampling blood from the lateral tail vein. Liver and lipid traits were measured using a Siemens ADVIA Chemistry XPT as described below.
  • An oral glucose tolerance test was administered upon an overnight fast (lasting 16 hours) at 20 weeks of age by administering 2 g/kg of body weight of Dextrose (Hospira Inc, NDC 0409-4902-34) by oral gavage.
  • Blood glucose was evaluated at defined time points (0, 15, 30, 60 and 120 minutes) using AlphaTrak blood glucose monitoring system (Zoetis U nited States, Parsippany NJ) by sampling blood from the lateral tail vein.
  • liver and lipid traits were measured using ADVIA Chemistry XPT System (Siemens Healthineers), an FDA approved clinical analyzer which we maintain and operate according to Siemens' guidelines.
  • the liver and lipid profile contains the following reagents: Alanine
  • ALT Aminotransferase
  • AST Aspartate Aminotransferase
  • AST Aspartate Aminotransferase
  • Cholesterol CHOL, Siemens REF 10376501
  • Direct H DL Cholesterol DHDL, Siemens REF 07511947
  • LDL Cholesterol Direct DLDL, Siemens REF 09793248
  • N EFA Wako 999-34691, 995-34791, 991-34891, 993-35191
  • Triglycerides Triglycerides
  • Explanted liver samples were fixed in 10% phosphate buffered formalin acetate at 4°C overnight, thoroughly rinsed in phosphate-buffered saline and embedded in paraffin wax.
  • For hematoxylin and eosin staining unstained 5 pm thick paraffin sections were deparaffinized in xylene then hydrated through graded alcohols up to water. Sections were stained with Carazzi's hematoxylin, washed in tap water, and then put in 95% ethanol. From there, they were put in eosin-phloxine solution then ran through graded alcohols to xylene. After xylene, the stained slides were cover-slipped and imaged stained and imaged using a 20X or 40X objective using the Aperio AT2 slide scanner (Leica Biosystems Inc.).
  • Liver protein was extracted using RIPA buffer (Cell signaling technology, Cat#9806) with Halt Protease & Phosphatase Inhibitor Cocktail (ThermoFisher Scientific, Cat#78440). Five microgram protein of each sample were separated in NuPAGE 4-12% Bis-Tris protein gel (Invitrogen, Cat#WG1403BOX), and transferred to nitrocellulose membrane using Trans-Blot ® TurboTM Transfer System (BioRad).
  • Blotting was performed using the following Cell Signaling Technology antibodies: Phospho-AMRKa (Thrl72) (Cell signaling technology, Cat#2535), AMPKa (Cell signaling technology, Cat#5831), b-Actin (Sigma, Cat#5441), Phospho-AKT (Ser473) (Cell signaling technology, Cat#4060), AKT (Cell signaling technology, Cat#9272), Phospho-LKBl (Ser428) (Cell signaling technology, Cat#3482), LKB1 (Cell signaling technology, Cat#3050), rabbit IgG conjugated to horseradish peroxidase (HRP) (Cell signaling technology, Cat#7074) and mouse IgG conjugated to HRP (Cell signaling technology, Cat#7076). Blots were developed using SuperSignal West Femto Substrate (ThermoFisher Scientific, Cat# 34095). Signals were captured using ImageQuant LAS4000 (GE Healthcare).
  • Example 15 Loss of Function Mutations in SLC39A5 Associate with Increasing Circulating Zinc Levels and Decreased T2D Risk

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KR1020217032315A KR20210139322A (ko) 2019-03-15 2020-03-12 용질 운반체 패밀리 39 구성원 5 (slc39a5) 억제제를 사용한 고혈당증의 치료
MX2021011015A MX2021011015A (es) 2019-03-15 2020-03-12 Tratamiento de la hiperglucemia con inhibidores del miembro 5 de la familia 39 de los transportadores de solutos (slc39a5).
EP20717451.7A EP3938513A2 (en) 2019-03-15 2020-03-12 Treatment of hyperglycemia with solute carrier family 39 member 5 (slc39a5) inhibitors
CN202080027032.4A CN113677368A (zh) 2019-03-15 2020-03-12 用溶质载体家族39成员5(slc39a5)抑制剂治疗高血糖症
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