Methods for treating skin cancer with histidine ammonia-lyase (HAL) agonists

Abstract

The present disclosure provides methods of treating a subject having or at risk of developing skin cancer, as well as methods of identifying subjects at increased risk of developing skin cancer.

Description

[Technical field]

[0001] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically as a text file of 197 kilobytes in size under the title 18923808302SEQ, created on July 2, 2022. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to the treatment of subjects having or at risk of developing skin cancer with histidine ammonia-lyase (HAL) agonists, and to methods of identifying subjects at increased risk of developing skin cancer. [Background technology]

[0003] Skin cancer refers to any cancer that occurs in the skin. These relatively common cancers are often mistaken by patients as non-malignant skin abnormalities, which can lead to delayed detection, difficult disease treatment, and potentially fatal outcomes. The most common type of skin cancer is basal cell carcinoma (BCC), which accounts for approximately 80% of all skin cancers. Other types of skin cancer include squamous cell carcinoma (SCC), which accounts for approximately 16% of all skin cancers, and melanoma, which accounts for approximately 4%. BCC and SCC are collectively referred to as non-melanoma skin cancer (NMSC). Melanoma is a metastatic cancer or carcinoma that develops from melanocytes in the epidermis, many of which are fatal. In 2000, it was reported that 47,000 people were identified as having new melanomas, of which 7,700 died (Non-Patent Document 1). It is estimated that melanoma caused by ultraviolet light is caused by intermittent exposure, such as intense sunburn, rather than chronic exposure to ultraviolet light (Non-Patent Document 2). Another rare form of invasive skin cancer is Merkel cell carcinoma (MCC), which resembles melanoma.

[0004] The HAL gene encodes histidine ammonia-lyase (or histidase), an enzyme that converts histidine (an essential amino acid that is incorporated into filaggrin, among other functions) to transurocanic acid, the major ultraviolet (UV)-absorbing chromophore that accumulates in the stratum corneum (Non-Patent Document 3). Inactivation of histidase is predicted to reduce the ability of the outermost layer of the epidermis to block UV light. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Greenlee et al.,Cancer J.Clin.,2000,50,7-33 [Non-Patent Document 2] Gilchrest et al., New Engl. J. Med., 1999, 340, 1341-1348 [Non-Patent Document 3] Barresi et al.,J.Invest.Dermatol.,2011,131,188-194 Summary of the Invention

[0006] The present disclosure provides a method of treating a subject having or at risk of developing skin cancer, the method comprising administering to the subject a HAL agonist. The disclosure also provides a method of treating a subject having non-melanoma skin cancer or preventing a subject from developing non-melanoma skin cancer, the method comprising administering to the subject a HAL agonist.

[0007] The present disclosure also provides a method of treating a subject having basal cell carcinoma or preventing a subject from developing basal cell carcinoma, the method comprising administering to the subject a HAL agonist.

[0008] The disclosure also provides a method of treating a subject having squamous cell carcinoma or preventing a subject from developing squamous cell carcinoma, the method comprising administering to the subject a HAL agonist.

[0009] The disclosure also provides a method of treating a subject having melanoma or preventing a subject from developing melanoma, the method comprising administering to the subject a HAL agonist. The present disclosure also provides a method of treating a subject having Merkel cell carcinoma or preventing a subject from developing Merkel cell carcinoma, the method comprising administering to the subject a HAL agonist.

[0010] The disclosure also provides a method of treating a subject having dermatofibrosarcoma protuberans or preventing a subject from developing dermatofibrosarcoma protuberans, the method comprising administering to the subject a HAL agonist.

[0011] The disclosure also provides a method of treating a subject having sebaceous gland carcinoma or preventing a subject from developing sebaceous gland carcinoma, the method comprising administering to the subject a HAL agonist. The disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing skin cancer, wherein the subject has or is at risk for developing skin cancer, the method comprising determining whether the subject has a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide by obtaining or obtaining a biological sample from the subject and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype that includes the HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide; treating or preventing skin cancer. administering or continuing to administer the therapeutic agent at a standard dosage to a subject who meets the HAL criteria, and / or administering a HAL agonist to the subject; administering or continuing to administer the therapeutic agent for treating or preventing skin cancer at the same or a lower standard dosage to a subject who is heterozygous for the HAL mutant nucleic acid molecule, and / or administering a HAL agonist to the subject; wherein the presence of a genotype having the HAL mutant nucleic acid molecule encoding a gain-of-function polypeptide predicted by the HAL indicates that the subject has a low risk of developing skin cancer.

[0012] The present disclosure also provides a method for identifying a subject having an elevated risk of developing skin cancer, the method comprising determining, or having determined, the presence or absence in a biological sample obtained from the subject of a HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL; wherein if the subject is HAL criterion, the subject has an elevated risk of developing skin cancer; and if the subject is heterozygous or homozygous for the HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL, the subject has a lower risk of developing skin cancer.

[0013] The disclosure also provides a therapeutic agent for treating or preventing skin cancer for use in treating or preventing skin cancer in a subject identified as having a genomic nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide, or its complement, wherein the genomic nucleic acid molecule has a nucleotide sequence that includes i) an adenine or its complement at a position corresponding to position 11,352 set forth in SEQ ID NO:2, or ii) a guanine or its complement at a position corresponding to position 14,441 set forth in SEQ ID NO:3.

[0014] The present disclosure also provides a HAL agonist for use in treating or preventing skin cancer in a subject, wherein the subject a) is reference for a HAL genomic nucleic acid molecule, a HAL mRNA molecule, or a HAL cDNA molecule, or b) is heterozygous for a genomic nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide, or its complement, wherein the genomic nucleic acid molecule has a nucleotide sequence that includes i) an adenine or its complement at a position corresponding to position 11,352 as set forth in SEQ ID NO:2, or ii) a guanine or its complement at a position corresponding to position 14,441 as set forth in SEQ ID NO:3.

[0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several features of the present disclosure. [Brief description of the drawings]

[0016] [Figure 1] Figure 1 shows the association between non-coding variants of HAL and gene expression in skin tissue (sun-exposed area - lower leg) from GTEx. [Diagram 2] 1 shows the association between non-coding variants in HAL and vitamin D levels and skin cancer-related traits. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Various terms relating to the aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other terms that are specifically defined are to be interpreted in a manner consistent with the definitions set forth herein.

[0018] Unless expressly stated otherwise, no method or embodiment set forth herein is intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically specifies in the claim or description that the steps are to be limited to a particular order, it is not intended to dictate order in any respect. This includes any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or workflow, general meanings derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0019] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. As used herein, the term "about" means that a cited numerical value is approximate, and small variations do not significantly affect the practice of the disclosed embodiments. When a numerical value is used, unless otherwise indicated by context, the term "about" means that the numerical value can vary by ±10% and remain within the range of the disclosed embodiments.

[0020] As used herein, the term "comprising" may in certain embodiments be replaced with "consisting" or "consisting essentially of," as desired.

[0021] As used herein, with respect to a nucleic acid molecule or polypeptide, the term "isolated" means that the nucleic acid molecule or polypeptide is in a state other than its native environment, e.g., away from blood and / or other tissues. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide can be in a highly purified form, i.e., greater than 95% pure or greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms.

[0022] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multistranded. A strand of a nucleic acid also refers to its complement.

[0023] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs), pet animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates (e.g., apes and monkeys). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0024] According to the present disclosure, a high frequency of putative gain of function (GOF) loads in the HAL gene associated with a reduced risk of developing skin cancer in humans has been identified. For example, it has been observed that a genetic mutation that changes the guanine at position 11,352 of the HAL reference genomic nucleic acid molecule (see SEQ ID NO: 1) to adenine or changes the adenine at position 14,441 of the HAL reference genomic nucleic acid molecule to guanine indicates that subjects with such mutations may have a lower risk of developing skin cancer. In summary, the genetic analysis described herein surprisingly shows that the HAL gene and, in particular, the pGOF of the HAL gene are associated with a reduced risk of developing skin cancer. Thus, subjects with HAL criteria at high risk of developing skin cancer, such as non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or sebaceous gland carcinoma, may be treated to prevent skin cancer, reduce symptoms, and / or inhibit the onset of symptoms. Thus, the present disclosure provides methods that utilize the identification of such variants in a subject to identify or stratify the risk in such a subject of developing skin cancer, such as non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or sebaceous gland carcinoma, or to diagnose a subject as having an elevated risk of developing skin cancer, such as non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or sebaceous gland carcinoma, such that subjects at risk or with active disease can be treated accordingly.

[0025] According to the present disclosure, it has been further observed that HAL variant nucleic acid molecules encoding HAL predicted gain-of-function polypeptides (regardless of whether these variants are homozygous or heterozygous in a particular subject) are associated with a reduced risk of developing skin cancer.Furthermore, the identification of the association of additional variants with gene burden masks according to the present disclosure indicates that HAL may be responsible for the protective effect in skin cancer.

[0026] For the purpose of this disclosure, any particular subject can be classified as having one of three HAL genotypes: i) HAL reference; ii) heterozygous for HAL variant nucleic acid molecule encoding HAL predicted gain-of-function polypeptide; or iii) homozygous for HAL variant nucleic acid molecule encoding HAL predicted gain-of-function polypeptide. If the subject does not have a copy of HAL variant nucleic acid molecule encoding HAL predicted gain-of-function polypeptide, the subject is HAL reference. If the subject has a single copy of HAL variant nucleic acid molecule, the subject is heterozygous for HAL variant nucleic acid molecule encoding HAL predicted gain-of-function polypeptide. As used herein, HAL variant nucleic acid molecule is any HAL nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that encodes a HAL polypeptide with partial gain-of-function, complete gain-of-function, predicted partial gain-of-function, or predicted complete gain-of-function. A subject having a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide having a partial gain-of-function (or predicted partial gain-of-function) is hypozygous for HAL. If a subject has two copies of a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide, the subject is homozygous for a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide.

[0027] For subjects who are genotyped or determined to be HAL-based, such subjects are at increased risk of developing skin cancer, such as non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, and / or sebaceous gland carcinoma. For subjects who are genotyped or determined to be HAL-based or heterozygous for a HAL variant nucleic acid molecule encoding a HAL-predicted gain-of-function polypeptide, such subjects can be treated with a HAL agonist.

[0028] In any of the embodiments described throughout this disclosure, the HAL mutant nucleic acid molecule can be any HAL nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a HAL polypeptide having a partial gain of function, a complete gain of function, a predicted partial gain of function, or a predicted complete gain of function.

[0029] In any of the embodiments described throughout this disclosure, the HAL predicted gain-of-function polypeptide can be any HAL polypeptide having a partial gain-of-function, a complete gain-of-function, a predicted partial gain-of-function, or a predicted complete gain-of-function.

[0030] Any one or more (i.e., any combination) of HAL variant nucleic acid molecules encoding HAL predicted gain-of-function polypeptides can be used in any of the methods described herein to determine whether a subject has an increased risk of developing skin cancer. A particular variant combination can form a mask that is used to statistically analyze a particular correlation between HAL and a reduced risk of developing skin cancer.

[0031] In any of the embodiments described throughout this disclosure, the skin cancer is non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or sebaceous gland carcinoma. In any of the embodiments described throughout this disclosure, the skin cancer is non-melanoma skin cancer. In any of the embodiments described throughout this disclosure, the skin cancer is basal cell carcinoma. In any of the embodiments described throughout this disclosure, the skin cancer is squamous cell carcinoma. In any of the embodiments described throughout this disclosure, the skin cancer is melanoma. In any of the embodiments described throughout this disclosure, the skin cancer is Merkel cell carcinoma. In any of the embodiments described throughout this disclosure, the skin cancer is dermatofibrosarcoma protuberans. In any of the embodiments described throughout this disclosure, the skin cancer is sebaceous gland carcinoma.

[0032] Symptoms of basal cell carcinoma include, but are not limited to, raised, smooth, pearly papules on sun-exposed skin of the individual's head, neck, or shoulders. Small blood vessels can often be seen within the tumor. Bleeding may occur as well as crusting of the tumor. Individuals may also mistake basal cell carcinoma for an ulcer that will not heal. Basal cell carcinoma is the least deadly form of skin cancer and can often be completely eradicated with proper treatment.

[0033] Symptoms of squamous cell carcinoma include, but are not limited to, scaly, thickened, red patches on an individual's sun-exposed skin. Some forms of squamous cell carcinoma appear as firm, hard lumps and dome shapes. Rupture and bleeding of the lumps may occur. If left untreated, squamous cell carcinoma may develop into a large mass. Squamous cell carcinoma is the second most common form of skin cancer.

[0034] Symptoms of melanoma include, but are not limited to, dark or brown to black lesions. Some melanomas appear pink, red, or flesh-colored and are called amelanotic melanomas. Amelanotic melanomas are a more aggressive form of melanoma. Some of the warning signs of malignant melanoma may include changes in size, shape, color, or height of moles, development of new moles during the transition from puberty to adulthood, itching, ulceration, or bleeding. Melanoma is the most deadly form of skin cancer.

[0035] Symptoms of Merkel cell carcinoma include, but are not limited to, fast-growing, non-tender flesh-colored to red / purple papules that are usually not painful or itchy. These papules appear on the skin of the head, neck, and arms that are highly sun-exposed. People often mistake Merkel cell carcinoma for a cyst or other type of cancer.

[0036] Symptoms of dermatofibrosarcoma protuberans include, but are not limited to, a small, slightly raised erythema or purpura of the skin, one to five centimeters wide, which may develop into a raised lump, which may in some cases cause redness, rupture, or bleeding.

[0037] Symptoms of sebaceous carcinoma include, but are not limited to, a slow-growing, sometimes yellow, painless lump on the eyelid. The papule may bleed or leak where the eyelid meets the eyelashes, and may develop a thickened, yellow or reddish crust.

[0038] The present disclosure provides a method of treating a subject having or at risk of developing skin cancer, the method comprising administering to the subject a HAL agonist. The disclosure also provides a method of treating a subject having or at risk of developing non-melanoma skin cancer, the method comprising administering to the subject a HAL agonist.

[0039] The present disclosure also provides a method of treating a subject having or at risk of developing basal cell carcinoma, the method comprising administering to the subject a HAL agonist. The present disclosure also provides a method of treating a subject having or at risk of developing squamous cell carcinoma, the method comprising administering to the subject a HAL agonist.

[0040] The present disclosure also provides a method of treating a subject having or at risk of developing melanoma, the method comprising administering to the subject a HAL agonist. The present disclosure also provides a method of treating a subject having or at risk of developing Merkel cell carcinoma, the method comprising administering to the subject a HAL agonist.

[0041] The disclosure also provides a method of treating a subject having or at risk of developing dermatofibrosarcoma protuberans, the method comprising administering to the subject a HAL agonist.

[0042] The disclosure also provides a method of treating a subject having or at risk of developing sebaceous gland carcinoma, the method comprising administering to the subject a HAL agonist. In some embodiments, the HAL agonist is a HAL protein, or a functionally active fragment thereof, or thyroid hormone (T3).

[0043] In some embodiments, the treatment method further comprises detecting the presence or absence of a HAL mutant nucleic acid molecule encoding a predicted gain-of-function polypeptide in a biological sample obtained from the subject. As used throughout this disclosure, a "HAL mutant nucleic acid molecule" is any HAL nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a HAL polypeptide having a partial gain-of-function, a complete gain-of-function, a predicted partial gain-of-function, or a predicted complete gain-of-function.

[0044] The present disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing skin cancer. In some embodiments, the subject has skin cancer. In some embodiments, the subject is at risk of developing skin cancer. In some embodiments, the method includes determining whether the subject has a HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL by obtaining or obtaining a biological sample from the subject and performing or performing a sequence analysis on the biological sample to determine whether the subject has a genotype that includes the HAL variant nucleic acid molecule. If the subject is HAL-based, the therapeutic agent for treating or preventing skin cancer is administered or continues to be administered to the subject at a standard dose, and / or a HAL agonist is administered to the subject. If the subject is heterozygous for the HAL variant, the therapeutic agent for treating or preventing skin cancer is administered or continues to be administered to the subject at the same or lower dose than the standard dose, and / or a HAL agonist is administered to the subject. The presence of the genotype with the HAL variant nucleic acid molecule that encodes the gain-of-function polypeptide predicted by HAL indicates that the subject has a low risk of developing skin cancer.In some embodiments, the subject is HAL-based.In some embodiments, the subject is heterozygous for the HAL variant nucleic acid molecule that encodes the gain-of-function polypeptide predicted by HAL.

[0045] For subjects who have been genotyped or determined to be heterozygous for a HAL variant nucleic acid molecule encoding a HAL-based or HAL-predicted gain-of-function polypeptide, such subjects can be treated with a HAL agonist as described herein.

[0046] Detecting the presence or absence of a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide in a biological sample obtained from a subject and / or determining whether a subject has a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide can be present in a cell obtained from the subject.

[0047] In some embodiments, if the subject is HAL-based, the subject is also administered a standard dose of a therapeutic agent for treating or preventing skin cancer.In some embodiments, if the subject is heterozygous for a HAL variant nucleic acid molecule that encodes a gain-of-function polypeptide predicted by HAL, the subject is administered a standard dose or a lower dose of a therapeutic agent for treating or preventing skin cancer.

[0048] In some embodiments, the method of treatment further comprises detecting the presence or absence of a HAL predicted gain-of-function polypeptide in a biological sample obtained from the subject. In some embodiments, if the subject does not have a HAL predicted gain-of-function polypeptide, the subject is administered a standard dose of a therapeutic agent for treating or preventing skin cancer. In some embodiments, if the subject has a HAL predicted gain-of-function polypeptide, the subject is administered a standard dose of a therapeutic agent for treating or preventing skin cancer. In some embodiments, if the subject has a HAL predicted gain-of-function polypeptide, the subject is administered a standard dose of a therapeutic agent for treating or preventing skin cancer.

[0049] The present disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing skin cancer. In some embodiments, the subject has skin cancer. In some embodiments, the subject is at risk of developing skin cancer. In some embodiments, the method includes determining whether the subject has a HAL predicted gain-of-function polypeptide by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a HAL predicted gain-of-function polypeptide. If the subject does not have a HAL predicted gain-of-function polypeptide, the therapeutic agent for treating or preventing skin cancer is administered or continues to be administered to the subject at a standard dose, and / or a HAL agonist is administered to the subject. If the subject has a HAL predicted gain-of-function polypeptide, the therapeutic agent for treating or preventing skin cancer is administered or continues to be administered to the subject at the same or lower dose than the standard dose, and / or a HAL agonist is administered to the subject. The presence of a HAL predicted gain-of-function polypeptide indicates that the subject is at low risk of developing skin cancer. In some embodiments, the subject has a HAL predicted gain-of-function polypeptide, hi some embodiments, the subject does not have a HAL predicted gain-of-function polypeptide.

[0050] Detecting the presence or absence of a HAL predicted gain-of-function polypeptide in a biological sample obtained from a subject and / or determining whether a subject has a HAL predicted gain-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the HAL predicted gain-of-function polypeptide can be present in a cell obtained from the subject.

[0051] Examples of therapeutic agents for treating or preventing skin cancer include thiazide diuretics (e.g., chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, or metolazone); potassium-sparing diuretics (e.g., amiloride, spironolactone, or triamterene); loop diuretics (e.g., bumetanide, furosemide, or torsemide); beta-blockers (e.g., acebutolol, atenolol, betaxolol, bisoprolol, bisoprolol / hydrochlorothiazide, metoprolol tartrate, metoprolol succinate, nadolol, pindolol, propranolol, solotol, or timolol); angiotensin-converting enzyme (ACE) agonists (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, or angiotensin II receptor blockers (ARBs) (e.g., candesartan, eprosartan, irbesartan, losartan, telmisartan, or valsartan); calcium channel blockers (e.g., amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nisoldipine, or verapamil); alpha blockers (e.g., doxazosin, prazosin, or terazosin); alpha beta blockers (e.g., carvedilol or labetalol); central agonists (e.g., methyldopa, clonidine, or guanfacine); vasodilators (e.g., hydralazine or minoxidil); aldosterone receptor antagonists (e.g., eplerenone or spironolactone), and renin agonists (e.g., aliskiren).

[0052] In some embodiments, the therapeutic agent for treating or preventing skin cancer is a thiazide diuretic, a potassium-sparing diuretic, a loop diuretic, a beta blocker, an ACE inhibitor, an ARB, a calcium channel blocker, an alpha blocker, an alpha-beta blocker, a central agonist, a vasodilator, an aldosterone receptor antagonist, or a renin inhibitor. In some embodiments, the thiazide diuretic is chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, or metolazone. In some embodiments, the potassium-sparing diuretic is amiloride, spironolactone, or triamterene. In some embodiments, the loop diuretic is bumetanide, furosemide, or torsemide. In some embodiments, the beta blocker is acebutolol, atenolol, betaxolol, bisoprolol, bisoprolol / hydrochlorothiazide, metoprolol tartrate, metoprolol succinate, nadolol, pindolol, propranolol, solotol, or timolol. In some embodiments, the ACE inhibitor is benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, or trandolapril. In some embodiments, the ARB is candesartan, eprosartan, irbesartan, losartan, telmisartan, or valsartan. In some embodiments, the calcium channel blocker is amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nisoldipine, or verapamil. In some embodiments, the alpha blocker is doxazosin, prazosin, or terazosin. In some embodiments, the alpha beta blocker is carvedilol or labetalol. In some embodiments, the central agonist is methyldopa, clonidine, or guanfacine. In some embodiments, the vasodilator is hydralazine or minoxidil. In some embodiments, the aldosterone receptor antagonist is eplerenone or spironolactone. In some embodiments, the renin inhibitor is aliskiren.

[0053] In some embodiments, the dose of a therapeutic agent for treating or preventing skin cancer can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% for a subject who is heterozygous for a HAL mutant nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL compared to a subject who is HAL-based (which may receive a standard dose). In some embodiments, the dose of a therapeutic agent for treating or preventing skin cancer can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dose of a therapeutic agent for treating or preventing skin cancer in a subject who is heterozygous for a HAL mutant nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL can be administered less frequently compared to a subject who is HAL-based.

[0054] The administration of the therapeutic agent and / or HAL agonist for treating or preventing skin cancer can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. The repeated administration can be the same dose or a different dose. The administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a particular dosing regimen, a subject can be treated for an extended period of time, such as, for example, 6 months, 1 year, or more. Furthermore, the therapeutic agent and / or HAL agonist for treating or preventing skin cancer can be administered sequentially or simultaneously. Furthermore, the therapeutic agent and / or HAL agonist for treating or preventing skin cancer can be administered in separate compositions or together in the same composition.

[0055] Administration of the therapeutic agent and / or HAL agonist for treating or preventing skin cancer can occur by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a single dose for administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharma-ceutical acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. The term "pharmaceutical acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially deleterious to the recipient thereof.

[0056] As used herein, the terms "treat", "treating", and "treatment" and "prevent", "prevention", and "prevention" refer to eliciting a desired biological response, such as a therapeutic effect and a preventative effect, respectively. In some embodiments, the therapeutic effect includes one or more of the following after administration of the agent or a composition comprising the agent: reduction / reduction of skin cancer, reduction / reduction in the severity of skin cancer (e.g., reduction or inhibition of onset of skin cancer), reduction / reduction in the severity of symptoms and skin cancer-related effects, delay in onset of symptoms and skin cancer-related effects, reduction in the severity of symptoms of skin cancer-related effects, reduction in the severity of acute episodes, reduction in the number of symptoms and skin cancer-related effects, reduction in the latency period of symptoms and skin cancer-related effects, amelioration of symptoms and skin cancer-related effects, reduction in secondary symptoms, reduction in secondary infections, prevention of recurrence of skin cancer, reduction in the number or frequency of recurrent episodes, increase in the latency period between symptomatic episodes, increase in the time to sustained progression, promotion of remission, induction of remission, enhancement of remission, acceleration of recovery, or increase in the effectiveness or reduction in resistance to alternative therapeutic agents, and / or increase in the survival time of the affected host animal. A prophylactic effect may include complete or partial avoidance / inhibition or delay (e.g., complete or partial avoidance / inhibition or delay) of the onset / progression of skin cancer following administration of a treatment protocol, and increasing the survival time of an affected host animal. Treating skin cancer includes treating a subject already diagnosed with some form of skin cancer, either at a clinical stage or clinical symptoms, delaying the onset or progression or progression or worsening of symptoms or signs of skin cancer, and / or preventing and / or reducing the severity of skin cancer.

[0057] The present disclosure also provides a method for identifying a subject at high risk of developing skin cancer. In some embodiments, the method includes determining or having determined the presence or absence of a HAL variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule and / or a cDNA molecule) encoding a HAL predicted gain-of-function polypeptide in a biological sample obtained from the subject. If the subject lacks a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide (i.e., the subject is classified as HAL-based by genotyping), the subject has a high risk of developing skin cancer. If the subject has a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide (i.e., the subject is heterozygous or homozygous for the HAL variant nucleic acid molecule), the subject has a lower risk of developing skin cancer compared to subjects who are HAL-based.

[0058] Having a single copy of a HAL mutant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide further protects a subject from developing skin cancer than not having a copy of a HAL mutant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide. Without intending to be limited to a particular theory or mechanism of action, it is believed that a single copy of a HAL mutant nucleic acid molecule (i.e., heterozygous for the HAL mutant nucleic acid molecule) protects a subject from developing skin cancer, and it is also believed that having two copies of a HAL mutant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide (i.e., homozygous for the HAL mutant nucleic acid molecule) may further protect a subject from developing skin cancer compared to a subject with a single copy. Thus, in some embodiments, a single copy of a HAL mutant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide may not completely protect a subject from developing skin cancer, but may instead provide partial or incomplete protection. Without wishing to be bound by any particular theory, there may be additional factors or molecules involved in the development of skin cancer that are still present in subjects having a single copy of a HAL mutant nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL, which may result in less than complete protection from the development of skin cancer.

[0059] Detecting the presence or absence of a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide in a biological sample obtained from a subject and / or determining whether a subject has a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide can be present in a cell obtained from the subject.

[0060] In some embodiments, once a subject is identified as having a high risk of developing skin cancer, the subject is further treated with a therapeutic agent and / or a HAL agonist for treating or preventing skin cancer, as described herein. For example, if a subject is HAL-based and therefore at high risk of developing skin cancer, the subject is administered a HAL agonist. In some embodiments, such a subject is also administered a therapeutic agent for treating or preventing skin cancer. In some embodiments, if the subject is heterozygous for a HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL, the subject is administered a therapeutic agent for treating or preventing skin cancer at a dose equal to or less than the standard dose, and / or is administered a HAL agonist. In some embodiments, the subject is HAL-based. In some embodiments, the subject is heterozygous for a HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL.

[0061] In some embodiments, any of the methods described herein may further comprise determining the total burden of the subject having a HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide and / or a HAL predicted gain-of-function variant polypeptide associated with a reduced risk of developing skin cancer. The total burden is the sum of all variants in the HAL gene, which may be performed in an association analysis with skin cancer. In some embodiments, the subject is homozygous for one or more of the HAL variant nucleic acid molecules encoding a HAL predicted gain-of-function polypeptide associated with a reduced risk of developing skin cancer. In some embodiments, the subject is heterozygous for one or more of the HAL variant nucleic acid molecules encoding a HAL predicted gain-of-function polypeptide associated with a reduced risk of developing skin cancer. The results of the association analysis suggest that the HAL variant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide is associated with a reduced risk of developing skin cancer. If the subject has a lower total load, the subject has a higher risk of developing skin cancer, and the subject is administered or continues to be administered with a standard dose of a therapeutic agent for treating or preventing skin cancer and / or a HAL agonist.If the subject has a higher total load, the subject has a lower risk of developing skin cancer, and the subject is administered or continues to be administered with a standard dose or a lower dose of a therapeutic agent for treating or preventing skin cancer.The higher the total load, the lower the risk of developing skin cancer.

[0062] HAL variants that can be used in the total load analysis include any one or more or any combination of those in Table 1 below.

[0063] [Table 1]

[0064] In some embodiments, the total load of subjects having any one or more HAL variant nucleic acid molecules encoding HAL predicted gain-of-function polypeptides represents the weighted sum of any multiple of the HAL variant nucleic acid molecules encoding HAL predicted gain-of-function polypeptides. In some embodiments, the total burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about 1,000,000 or more genetic variants present in or surrounding (up to 10 Mb) the HAL gene, where the genetic burden is the number of alleles multiplied by the estimated association with skin cancer or a related outcome for each allele (e.g., a weighted polygenic burden score). This can include any genetic variants that are close to the HAL gene (up to 10Mb around the gene) that show non-zero association with skin cancer-related traits in gene association analysis, regardless of genome annotation.In some embodiments, if a subject has a total load higher than a desired threshold score, the subject has a low risk of developing skin cancer.In some embodiments, if a subject has a total load lower than a desired threshold score, the subject has a high risk of developing skin cancer.

[0065] In some embodiments, the total burden can be divided into quintiles, for example, top quintile, middle quintile, and bottom quintile, with the top quintile of total burden corresponding to the lowest risk group and the bottom quintile of total burden corresponding to the highest risk group. In some embodiments, subjects with a larger total burden include the highest weighted total burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% total burden from the subject population. In some embodiments, the genetic variants include genetic variants that have an association with skin cancer in the top 10%, top 20%, top 30%, top 40%, or top 50% of the p-value range for the association. In some embodiments, each of the identified genetic variants is about 10 -2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 , about 10 -7 , about 10 -8 , about 10 -9 , about 10 -10 , about 10 -11 , about 10 -12 , about 10 -13 , about 10 -14 , or about 10 -15 In some embodiments, the identified genetic variants include those with an association with skin cancer at a p-value of 5×10 -8In some embodiments, the identified genetic variants include genetic variants that are associated with skin cancer at a p-value of less than about 1.5, 1.75, 2.0, or 2.25 for the top 20% of the distribution; or genetic variants that are associated with skin cancer in subjects at high risk compared to the remainder of a reference population with an odds ratio (OR) of about 1.5 or more, about 1.75 or more, about 2.0 or more, about 2.25 or more, about 2.5 or more, or about 2.75 or more. In some embodiments, the odds ratio (OR) may range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or may be greater than 7.0. In some embodiments, high-risk subjects include subjects with a total burden in the bottom decile, quintile, or tertile in the reference population. The total burden threshold is determined based on the nature of the intended practical application and the risk difference that is considered meaningful for that practical application.

[0066] In some embodiments, once a subject is identified as having a high risk of developing skin cancer, the subject is further administered a therapeutic agent and / or a HAL agonist for treating or preventing skin cancer, as described herein. For example, if a subject is HAL-based and therefore at high risk of developing skin cancer, the subject is administered a HAL agonist. In some embodiments, such a subject is also administered a therapeutic agent for treating or preventing skin cancer. In some embodiments, if the subject is heterozygous for a HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL, the subject is administered a therapeutic agent for treating or preventing skin cancer at a dose equal to or less than the standard dose, and / or is administered a HAL agonist. In some embodiments, the subject is HAL-based. In some embodiments, the subject is heterozygous for a HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL. Furthermore, if a subject has a lower total burden of HAL variant nucleic acid molecules encoding gain-of-function polypeptides predicted by HAL, and therefore has a higher risk of developing skin cancer, the subject is administered a therapeutic agent for treating or preventing skin cancer. In some embodiments, if a subject has a lower total burden of HAL variant nucleic acid molecules encoding gain-of-function polypeptides predicted by HAL, the subject is administered a therapeutic agent for treating or preventing skin cancer at a dose equal to or higher than the standard dose administered to a subject with a higher total burden of HAL variant nucleic acid molecules encoding gain-of-function polypeptides predicted by HAL.

[0067] The present disclosure also provides a method for detecting the presence or absence of a HAL mutant genomic nucleic acid molecule that encodes a gain-of-function polypeptide predicted by HAL in a biological sample obtained from a subject. It is understood that gene sequences in a population, and the mRNA molecules encoded by such genes, may differ due to polymorphisms, such as single nucleotide polymorphisms (SNPs). The sequences provided herein for HAL mutant genomic nucleic acid molecules are merely exemplary sequences. Other sequences for HAL mutant genomic nucleic acid molecules are also possible.

[0068] The biological sample can be derived from any cell, tissue, or biological fluid of a subject. The biological sample may include any clinically relevant tissue, such as, for example, a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of a bodily fluid, such as, for example, blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, the biological sample includes a buccal swab. The biological sample used in the methods disclosed herein may vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample may be subjected to different treatments depending on the assay employed. For example, when detecting HAL mutant nucleic acid molecules, a pretreatment designed to isolate or enrich the biological sample for HAL mutant nucleic acid molecules may be employed. For this purpose, various techniques may be used. When detecting the level of HAL mutant mRNA molecules, various techniques may be used to enrich the biological sample containing mRNA molecules. Various methods may be used to detect the presence or level of mRNA molecules, or the presence of a particular mutant genomic DNA locus.

[0069] The disclosure also provides a method for detecting a HAL mutant nucleic acid molecule or its complement encoding a HAL predicted gain-of-function polypeptide in a subject, the method comprising assaying a biological sample obtained from the subject to determine whether a nucleic acid molecule in the biological sample is a HAL mutant nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide.

[0070] In some embodiments, the HAL mutant nucleic acid molecule encoding a predicted HAL gain-of-function polypeptide, or its complement, is a genomic nucleic acid molecule having a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 11,352 of SEQ ID NO:2; or a guanine or its complement at a position corresponding to position 14,441 of SEQ ID NO:3. In some embodiments, the HAL mutant genomic nucleic acid molecule encoding a predicted HAL gain-of-function polypeptide, or its complement, has a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 11,352 of SEQ ID NO:2. In some embodiments, the HAL mutant genomic nucleic acid molecule encoding a predicted HAL gain-of-function polypeptide, or its complement, has a nucleotide sequence that includes a guanine or its complement at a position corresponding to position 14,441 of SEQ ID NO:3.

[0071] In some embodiments, the biological sample comprises cells or cell lysates. Such methods can further comprise, for example, obtaining a biological sample from a subject that comprises a HAL genomic nucleic acid molecule. Such assays can comprise, for example, determining the identity of these positions of a particular HAL nucleic acid molecule. In some embodiments, the methods are in vitro methods.

[0072] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of the HAL nucleic acid molecule or its complement in the biological sample. In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of the HAL genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises: a position corresponding to position 11,352 in SEQ ID NO:2 or its complement, or a position corresponding to position 14,441 in SEQ ID NO:3 or its complement. If the sequenced portion of the HAL nucleic acid molecule in the biological sample comprises an adenine or its complement at the position corresponding to position 11,352 in SEQ ID NO:2 or a guanine or its complement at the position corresponding to position 14,441 in SEQ ID NO:3, the HAL nucleic acid molecule in the biological sample is a HAL mutant nucleic acid molecule encoding a gain-of-function polypeptide predicted for HAL.

[0073] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a HAL genomic nucleic acid molecule or its complement that is adjacent to a position corresponding to position 11,352 set forth in SEQ ID NO:2 or its complement, or adjacent to a position corresponding to position 14,441 set forth in SEQ ID NO:3 or its complement; b) extending the primer at least through a position in the nucleotide sequence of a HAL genomic nucleic acid molecule or its complement that is adjacent to a position corresponding to position 11,352 set forth in SEQ ID NO:2 or its complement, or corresponding to a position corresponding to position 14,441 set forth in SEQ ID NO:3 or its complement; and c) determining whether the extension product of the primer comprises: an adenine or its complement at the position corresponding to position 11,352 set forth in SEQ ID NO:2, or a guanine or its complement at the position corresponding to position 14,441 set forth in SEQ ID NO:3.

[0074] In some embodiments, the assay involves sequencing the entire nucleic acid molecule, hi some embodiments, only the HAL genomic nucleic acid molecule is analyzed. In some embodiments, the assay comprises: a) amplifying at least a portion of a HAL nucleic acid molecule or its complement in a biological sample, wherein the amplified portion comprises: an adenine or its complement at a position corresponding to position 11,352 set forth in SEQ ID NO:2 or a guanine or its complement at a position corresponding to position 14,441 set forth in SEQ ID NO:3; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising an adenine or its complement at a position corresponding to position 11,352 set forth in SEQ ID NO:2 or a guanine or its complement at a position corresponding to position 14,441 set forth in SEQ ID NO:3; and d) detecting the detectable label.

[0075] In some embodiments, the assay comprises: a) amplifying at least a portion of a HAL genomic nucleic acid molecule or its complement in a biological sample, the portion comprising: an adenine or its complement at a position corresponding to position 11,352 set forth in SEQ ID NO:2, or a guanine or its complement at a position corresponding to position 14,441 set forth in SEQ ID NO:3; and b) detecting a detectable label.

[0076] In some embodiments, the assay includes contacting a HAL nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the HAL nucleic acid molecule or its complement comprising an adenine or its complement at a position corresponding to position 11,352 set forth in SEQ ID NO:2 or a guanine or its complement at a position corresponding to position 14,441 set forth in SEQ ID NO:3; and detecting the detectable label.

[0077] In some embodiments, the HAL nucleic acid molecule is present in a cell obtained from the subject. Mutation-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in nucleotide sequences. Mutation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0078] In some embodiments, the assay involves RNA sequencing (RNA-Seq). In some embodiments, the assay also involves reverse transcribing mRNA into cDNA, for example, by reverse transcription polymerase chain reaction (RT-PCR).

[0079] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or identify the polynucleotides comprising the HAL variant genomic nucleic acid molecule. The hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length that is sufficient for use in the detection method of choice, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under highly stringent hybridization conditions. The probes and primers can have complete nucleotide sequence identity to consecutive nucleotides in the target nucleotide sequence, although probes that are different from the target nucleotide sequence and that retain the ability to specifically detect and / or identify the target nucleotide sequence can be designed by conventional methods. The 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 to the nucleotide sequence of the target nucleic acid molecule.

[0080] In some embodiments, to determine whether a HAL nucleic acid molecule (e.g., a genomic nucleic acid molecule) or its complement in a biological sample contains a nucleotide sequence or its complement that contains an adenine at a position corresponding to position 11,352 of SEQ ID NO:2, 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 the adenine at a position corresponding to position 11,352 of SEQ ID NO:2 and a second primer derived from the 3' flanking sequence adjacent to the adenine at a position corresponding to position 11,352 of SEQ ID NO:2 to generate an amplicon that indicates the presence of a SNP at a position that codes for an adenine at a position corresponding to position 11,352 of SEQ ID NO:2. In some embodiments, the length of the amplicon can range from a combination of the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be generated by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region including an adenine at a position corresponding to position 11,352 in SEQ ID NO:2 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position including an adenine at a position corresponding to position 11,352 in SEQ ID NO:2.

[0081] In some embodiments, to determine whether a HAL nucleic acid molecule (e.g., a genomic nucleic acid molecule) or its complement in a biological sample contains a nucleotide sequence containing a guanine at a position corresponding to position 14,441 of SEQ ID NO:3 or its complement, the biological sample can be subjected to an amplification method using a primer pair including a first primer derived from a 5' flanking sequence adjacent to the guanine at a position corresponding to position 14,441 of SEQ ID NO:3 and a second primer derived from a 3' flanking sequence adjacent to the guanine at a position corresponding to position 14,441 of SEQ ID NO:3 to generate an amplicon indicating the presence of a SNP at a position encoding a guanine at a position corresponding to position 14,441 of SEQ ID NO:3. In some embodiments, the length of the amplicon can range from a combination of the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be generated by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region including a position that includes a guanine at position corresponding to 14,431 in SEQ ID NO:3 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of a position that includes a guanine at position corresponding to 14,441 in SEQ ID NO:3.

[0082] Similar amplicons can be generated from mRNA and / or cDNA sequences. PCR primer pairs can be derived from known sequences, for example, using computer programs designed for that purpose, such as the PCR primer analysis tools 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, sequences can be visually inspected and primers manually specified using known guidelines.

[0083] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing (fluorescence in situ hybridization (FISH)), including the use of labeled primers or labeled probes on purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule may be amplified prior to or simultaneously with detection. Illustrative examples of 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).

[0084] Hybridization techniques can employ stringent conditions so that the probe or primer specifically hybridizes with its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other non-target sequences, for example, at least 2-fold, at least 3-fold, at least 4-fold or more above background, including more than 10-fold above background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than other nucleotide sequences, more than 10-fold above background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0085] Suitable stringent conditions that promote DNA hybridization, such as 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by a 2x SSC wash at 50°C, are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection include conditions in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. +ion, usually about 0.01 to 1.0 M Na + The conditions will be ionic concentration (or other salts) and temperature of at least about 30° C. for short probes (e.g., 10-50 nucleotides) and at least about 60° C. for longer probes (e.g., more than 50 nucleotides). Stringent conditions may be achieved by the addition of destabilizing agents such as formamide. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash will be at least long enough to reach equilibrium.

[0086] The present disclosure also provides a method for detecting the presence of a predicted gain-of-function polypeptide in HAL, comprising performing an assay on a biological sample obtained from a subject to determine whether the HAL polypeptide in the biological sample contains one or more mutations that cause the polypeptide to have a gain-of-function (partial or complete) or a predicted gain-of-function (partial or complete).

[0087] In some embodiments, if a subject does not have a HAL predicted gain-of-function polypeptide, the subject has a high risk of developing skin cancer, such as non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or sebaceous gland carcinoma. In some embodiments, if a subject has a HAL predicted gain-of-function polypeptide, the subject has a low risk of developing skin cancer, such as non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or sebaceous gland carcinoma.

[0088] The present disclosure also provides an isolated nucleic acid molecule that hybridizes with HAL mutant genomic nucleic acid molecule.In some embodiments, such isolated nucleic acid molecule hybridizes with HAL mutant nucleic acid molecule under stringent conditions.Such nucleic acid molecule can be used as, for example, probe, primer, mutation-specific probe or mutation-specific primer as described or exemplified herein.

[0089] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a HAL mutant nucleic acid molecule that includes a position corresponding to position 11,352 set forth in SEQ ID NO:2 or position 14,441 set forth in SEQ ID NO:3.

[0090] In some embodiments, such isolated nucleic acid molecules comprise at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55 , at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 18 to about 30 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0091] In some embodiments, the isolated mutation-specific probe or primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or primer comprises a nucleotide sequence that is complementary to a nucleotide sequence of a portion of a HAL mutant nucleic acid molecule encoding a predicted gain-of-function polypeptide in HAL or its complement, in some embodiments, the portion comprises a position corresponding to position 11,352 or its complement as set forth in SEQ ID NO:2, or position 14,441 or its complement as set forth in SEQ ID NO:3.

[0092] In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a HAL variant genomic nucleic acid molecule. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 35 nucleotides.

[0093] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of a HAL mutant nucleic acid molecule that encodes a predicted gain-of-function polypeptide in HAL, the portion comprising a position corresponding to position 11,352 or its complement set forth in SEQ ID NO:2, or position 14,441 or its complement set forth in SEQ ID NO:3.

[0094] In some embodiments, the mutation-specific probe and the mutation-specific primer comprise DNA. In some embodiments, the mutation-specific probe and the mutation-specific primer comprise RNA.

[0095] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein or their complements. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0096] In some embodiments, the primers can be used in second generation or high throughput sequencing, including mutation specific primers. Sometimes the primers can be modified, including mutation specific primers. In particular, the primers can include various modifications used in various steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including biotinylated primers in the cloning step, and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is typically performed using paired-end tag libraries, where each molecule of DNA template is about 135 bp in length. Biotinylated primers are used in the bead loading and emulsion PCR steps. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. The adapters can contain 5'-biotin tags for immobilizing the DNA library on streptavidin-coated beads.

[0097] The probes and primers described herein can be used to detect nucleotide variations in any of the HAL mutant genomic nucleic acid molecules disclosed herein.The primers described herein can be used to amplify the HAL mutant genomic nucleic acid molecules or fragments thereof.

[0098] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with guanine (rather than adenine) at a position corresponding to position 11,352 of SEQ ID NO:1 in a particular HAL nucleic acid molecule, the presence of an amplified fragment indicates the presence of a HAL reference genomic nucleic acid molecule. Conversely, if one of the 3' ends of the primer hybridizes with adenine (rather than guanine) at a position corresponding to position 11,352 of SEQ ID NO:2 in a particular HAL nucleic acid molecule, the presence of an amplified fragment indicates the presence of a HAL variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer that is complementary to the adenine at a position corresponding to position 11,352 of SEQ ID NO:2 can be at the 3' end of the primer.

[0099] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with adenine (rather than guanine) at a position corresponding to position 14,441 of SEQ ID NO:1 in a particular HAL nucleic acid molecule, the presence of an amplified fragment indicates the presence of a HAL reference genomic nucleic acid molecule. Conversely, if one of the 3' ends of the primer hybridizes with guanine (rather than adenine) at a position corresponding to position 14,441 of SEQ ID NO:3 in a particular HAL nucleic acid molecule, the presence of an amplified fragment indicates the presence of a HAL variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer that is complementary to the guanine at a position corresponding to position 14,441 of SEQ ID NO:3 can be at the 3' end of the primer.

[0100] In the context of this disclosure, "specifically hybridizes" means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer) does not hybridize to a nucleotide sequence encoding a genomic nucleic acid molecule of the HAL reference.

[0101] In any of the embodiments described throughout this disclosure, the probe (e.g., the mutation-specific probe) can include a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0102] The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid-state substrate or support to which a molecule, such as any of the probes disclosed herein, can associate. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which a plurality of different probes are attached in an array, grid, or other organized pattern. A form of solid-state substrate is a microtiter dish, such as a standard 96-well format. In some embodiments, a multi-well glass slide can be used, usually containing one array per well. In some embodiments, the support is a microarray.

[0103] The present disclosure also provides a molecular complex comprising or consisting of any of the HAL mutant genomic nucleic acid molecules described herein, or their complements, and any of the mutation-specific primers or mutation-specific probes described herein. In some embodiments, the HAL mutant genomic nucleic acid molecules in the molecular complex, or their complements, are single-stranded. In some embodiments, the molecular complex comprises or consists of any of the HAL mutant genomic nucleic acid molecules described herein, or their complements, and any of the mutation-specific primers described herein. In some embodiments, the molecular complex comprises or consists of any of the HAL mutant genomic nucleic acid molecules described herein, or their complements, and any of the mutation-specific probes described herein.

[0104] In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to a HAL genomic nucleic acid molecule encoding a predicted gain-of-function polypeptide in HAL, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the HAL genomic nucleic acid molecule at a position corresponding to position 11,352 set forth in SEQ ID NO:2 or its complement.

[0105] In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to a HAL genomic nucleic acid molecule encoding a predicted gain-of-function polypeptide in HAL, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the HAL genomic nucleic acid molecule at a position corresponding to position 14,441 set forth in SEQ ID NO:3 or its complement.

[0106] In some embodiments, the genomic nucleic acid molecule of the molecular complex comprises SEQ ID NO: 2. In some embodiments, the genomic nucleic acid molecule of the molecular complex comprises SEQ ID NO: 3. In some embodiments, the molecular complex comprises a mutation-specific probe or a mutation-specific primer that comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin. In some embodiments, the molecular complex further comprises a non-human polymerase.

[0107] The nucleotide sequence of the HAL reference genome nucleic acid molecule is set forth in SEQ ID NO:1 (ENSG00000084110.11, encompassing chr12:95,972,662-95,996,344 in the GRCh38 / hg38 human genome assembly). With reference to SEQ ID NO:1, position 11,352 is a guanine. With reference to SEQ ID NO:1, position 14,441 is an adenine.

[0108] There exists a HAL mutant genomic nucleic acid molecule in which the guanine at position 11,352 is substituted with an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:2 (rs3819817).

[0109] Another HAL mutant genomic nucleic acid molecule exists in which the adenines at positions 14,41 are replaced with guanines, the nucleotide sequence of which is set forth in SEQ ID NO:3 (rs10859995).

[0110] The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:4. The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:5. The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:6. The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:7. The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:8. The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:9. The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:10. The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:11. The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:12. The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:13. The nucleotide sequence of another HAL reference mRNA molecule is set forth in SEQ ID NO:14.

[0111] The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 15. The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 16. The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 17. The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 18. The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 19. The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 20. The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 21. The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 22. The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 23. The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 24. The nucleotide sequence of another HAL Reference cDNA molecule is set forth in SEQ ID NO: 25.

[0112] The genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be derived from any organism. For example, the genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be an ortholog from human or another organism (e.g., non-human mammal, rodent, mouse, or rat). It is understood that gene sequences within a population can differ due to polymorphisms, such as single nucleotide polymorphisms. The examples provided herein are only exemplary sequences. Other sequences are also possible.

[0113] Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Functional polynucleotides can act as effectors, agonists, modulators, and stimulators of the specific activity of target molecules, or functional polynucleotides can have de novo activity independent of any other molecules.

[0114] The isolated nucleic acid molecules disclosed herein can include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to heterologous nucleic acid sequences, for example in a vector, or heterologous labels. For example, the isolated nucleic acid molecules disclosed herein can be present as exogenous donor sequences in or containing a vector that includes the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to heterologous labels. The labels can be directly detectable (e.g., fluorophores) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The labels can also be, for example, chemiluminescent materials; metal-containing materials; or enzymes, where enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent and chemiluminescent substrates, as well as other labels.

[0115] The isolated nucleic acid molecule or its complement can be present in a host cell. In some embodiments, the host cell can comprise a vector comprising any of the nucleic acid molecules described herein or their complements. In some embodiments, the nucleic acid molecule is operably linked to a promoter active in the host cell. In some embodiments, the promoter is an exogenous promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a mammalian cell.

[0116] The disclosed nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0117] The nucleic acid molecules disclosed herein may also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains modifications to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (cytosine), and thymine. Examples of uracils and cytosines include, but are not limited to, 4-isopropyl uracil, 4-isopropyl uracil, 8 ...

[0118] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C 2~10Exemplary 2' sugar modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n Other modifications at the 2' position include, but are not limited to, C 1~10 Examples of suitable substituents include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications may be made at other positions on the sugar, particularly the 3' position of the sugar in the 3' terminal nucleotide or 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0119] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain reverse polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0120] The present disclosure also provides a vector comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector can be a viral vector or a non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (such as a circular double stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YACs), Epstein-Barr (EBV) derived episomes, and other expression vectors known in the art.

[0121] Desirable regulatory sequences for mammalian host cell expression may include, for example, viral elements directing high levels of polypeptide expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma derived promoters and / or enhancers, as well as strong mammalian promoters, such as native immunoglobulin promoters and actin promoters. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known. The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter).

[0122] Percent identity (%) (or percent complementarity) between specific stretches of nucleotide sequences in nucleic acid molecules or amino acid sequences in polypeptides can be routinely determined using the BLAST program (basic sequence comparison search tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings that use the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). When referring to percent sequence identity in this specification, a higher percent sequence identity is preferred over a lower one.

[0123] The present disclosure also provides a composition comprising one or more of the isolated nucleic acid molecules, genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules disclosed herein, or a vector comprising the same. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution, such as PBS, HBSS, and the like.

[0124] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of numbering a particular nucleotide or sequence of nucleotides or position, refers to the numbering of a specified reference sequence when the particular nucleotide or sequence of nucleotides is compared to a reference sequence (e.g., SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3). In other words, the residue (e.g., nucleotide or amino acid) number or residue (e.g., nucleotide or amino acid) position of a particular polymer is specified with reference to a reference sequence, not by the actual position number of the residue within the particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps exist, the numbering of the residues in the particular nucleotide or nucleotide sequence is done with reference to the reference sequence to which it is aligned.

[0125] For example, a HAL variant genomic nucleic acid molecule comprising a nucleotide sequence encoding a predicted gain-of-function polypeptide for HAL, which nucleotide sequence comprises an adenine at position corresponding to position 11,352 set forth in SEQ ID NO:2, means that when the nucleotide sequence of the HAL genomic nucleic acid molecule is aligned against the sequence of SEQ ID NO:2, the sequence of HAL has an adenine residue at position corresponding to position 11,352 of SEQ ID NO:2. These terms refer to a HAL variant genomic nucleic acid molecule that encodes a predicted gain-of-function polypeptide for HAL, wherein the genomic nucleic acid molecule has a nucleotide sequence that comprises an adenine residue that is homologous to the adenine residue at position 11,352 of SEQ ID NO:2.

[0126] As described herein, the position in the HAL variant genomic nucleic acid molecule corresponding to position 11,352 of SEQ ID NO:2 can be identified, for example, by performing sequence comparison between the nucleotide sequence of a particular HAL genomic nucleic acid molecule and the nucleotide sequence of SEQ ID NO:2. For example, there are various computer algorithms that can be used to perform sequence comparison to identify the position of the nucleotide corresponding to position 11,352 of SEQ ID NO:2. For example, sequence comparison can be performed by using the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res., 1997, 25, 3389-3402) or CLUSTALW software (Sievers and Higgins, Methods Mol. Biol., 2014, 1079, 105-116). However, sequences can also be aligned manually.

[0127] The amino acid sequence of the HAL reference polypeptide is set forth in SEQ ID NO: 26. With reference to SEQ ID NO: 26, the HAL reference polypeptide is 657 amino acids in length. The amino acid sequence of another HAL reference polypeptide is set forth in SEQ ID NO: 27. With reference to SEQ ID NO: 27, the HAL reference polypeptide is 449 amino acids in length.

[0128] The amino acid sequence of another HAL reference polypeptide is set forth in SEQ ID NO: 28. With reference to SEQ ID NO: 28, the HAL reference polypeptide is 591 amino acids in length. The amino acid sequence of another HAL reference polypeptide is set forth in SEQ ID NO: 29. With reference to SEQ ID NO: 29, the HAL reference polypeptide is 219 amino acids in length.

[0129] The amino acid sequence of another HAL reference polypeptide is set forth in SEQ ID NO: 30. With reference to SEQ ID NO: 30, the HAL reference polypeptide is 167 amino acids in length. Because the HAL mutant nucleic acid molecules described herein contain mutations within the non-coding regions of HAL, the predicted gain-of-function polypeptide for HAL can be any of the aforementioned HAL polypeptides encoded by any of the HAL mutant nucleic acid molecules described herein.

[0130] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-end of the sequence and proceeding toward the 3'-end (i.e., from left to right in each sequence). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by reference to the shown strand. The amino acid sequences follow the standard convention of beginning at the amino-terminus of the sequence and proceeding toward the carboxy-terminus (i.e., from left to right in each sequence).

[0131] The present disclosure also provides a therapeutic agent for treating or preventing skin cancer for use in treating or preventing skin cancer in a subject (or for use in preparing a medicament for treating or preventing skin cancer), wherein the subject has any of the HAL mutant genomic nucleic acid molecules encoding the HAL predicted gain-of-function polypeptides described herein. The therapeutic agent for treating or preventing skin cancer can be any of the therapeutic agents for treating or preventing skin cancer described herein. The skin cancer can be any of non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, and sebaceous carcinoma.

[0132] In some embodiments, the subject is identified as having a genomic nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide having a nucleotide sequence including an adenine at a position corresponding to position 11,352 set forth in SEQ ID NO:2, or its complement.

[0133] In some embodiments, the subject is identified as having a genomic nucleic acid molecule encoding a HAL predicted gain-of-function polypeptide having a nucleotide sequence including a guanine or its complement at a position corresponding to position 14,441 set forth in SEQ ID NO:3, or its complement.

[0134] The present disclosure also provides a HAL agonist for use in treating or preventing skin cancer in a subject (or for use in preparing a medicament for treating or preventing skin cancer), wherein the subject is heterozygous for any of the HAL variant genomic nucleic acid molecules encoding the HAL predicted gain-of-function polypeptides described herein, or the subject is a reference for the HAL genomic nucleic acid molecule. The HAL agonist can be any of the HAL agonists described herein. The skin cancer can be any of non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, and sebaceous carcinoma.

[0135] In some embodiments, the subject is a reference for a HAL genomic nucleic acid molecule. In some embodiments, the subject is heterozygous for a genomic nucleic acid molecule encoding a HAL-predicted gain-of-function polypeptide having a nucleotide sequence including an adenine at a position corresponding to position 11,352 set forth in SEQ ID NO:2, or its complement.

[0136] In some embodiments, the subject is heterozygous for a genomic nucleic acid molecule encoding a HAL-predicted gain-of-function polypeptide having a nucleotide sequence including a guanine at a position corresponding to position 14,441 set forth in SEQ ID NO:3, or its complement.

[0137] All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual document was specifically and individually indicated to be so incorporated by reference. Where various versions of a sequence are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date or the filing date of the priority application to which the accession number refers, if applicable. Similarly, where different versions of publications, websites, etc. have been published at different times, the version last published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure may be used in combination with any other feature, step, element, embodiment, or aspect, unless otherwise indicated. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0138] The following examples are provided to further illustrate the embodiments. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide those skilled in the art with a disclosure and explanation of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated, and are intended to be merely illustrative and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation can be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0139] Example 1: Association of HAL GOF Mutations with Skin Cancer The exomes of 454,787 UKB study participants were sequenced, covering 95.8% of the target bases at a depth of more than 20x, as previously described (Szustakowski, Advancing Human Genetics Research and Drug Discovery through Exome Sequencing of the UK Biobank. bioRxiv, 2021; and Van Hout et al., Nature, 2020). 12 million variants were identified in 39 million base pairs across the coding regions of 18,659 genes (data not shown). Among the variants identified, there were 3,375,252 synonymous variants (median 10,260 per individual), 7,689,495 missense variants (9,284 per individual), and 889,957 putative gain-of-function (pLOF) variants (212 per individual) (data not shown), of which approximately half were observed only once in this dataset (singleton variants; data not shown).

[0140] We found that rare pLOF and deleterious missense variant burden in HAL was associated not only with higher vitamin D levels, but also with increased susceptibility to sunburn and an increased risk of actinic keratosis and non-melanoma skin cancer (Figure 2). These findings independently colocalize with an expression quantification trait locus (rs3819817:T) that increases HAL expression in skin tissue (r 2 =0.97), supported by a trait-reducing association with a high-frequency variant (rs10859995:C, 58% frequency) (GT Consortium, Science, 2020, 369, 1318-1330; see Figure 1). Collectively, these results link HAL to both vitamin D levels and skin cancer and reveal an allelic lineage that contains a high frequency of gain-of-function noncoding variants (trait-reducing).

[0141] In addition to those described herein, various modifications of the described subject matter will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety and for all purposes.

Claims

A method in vitro for determining the susceptibility of a subject to developing skin cancer, the method comprising: determining, or having determined, the presence or absence of a HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by histidine ammonia-lyase (HAL) in a biological sample obtained from the subject; wherein when the subject is a HAL reference, the subject is represented as having a high risk of developing skin cancer; wherein when the subject is heterozygous or homozygous for a HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by the HAL, the subject is represented as having a low risk of developing skin cancer, said method. **Claim 2** The method according to claim 1, wherein the HAL variant nucleic acid molecule is a genomic nucleic acid molecule having a nucleotide sequence comprising adenine at a position corresponding to position 11,352 as set forth in SEQ ID NO: 2 or guanine at a position corresponding to position 14,441 as set forth in SEQ ID NO:

3. **Claim 3** Use of a therapeutic agent for treating or preventing skin cancer in the manufacture of a medicament for treating or preventing skin cancer in a subject who is heterozygous or homozygous for a HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by histidine ammonia-lyase (HAL). **Claim 4** Use of a histidine ammonia-lyase (HAL) agonist in the manufacture of a medicament for treating or preventing skin cancer in a subject, wherein the subject is a) a reference for a HAL genomic nucleic acid molecule, or b) heterozygous for a genomic nucleic acid molecule encoding a gain-of-function polypeptide predicted by HAL or its complement, said use.

5. The use according to claim 4, wherein the HAL agonist is a HAL protein or a thyroid hormone (T ３ ). **Claim 6** The method according to claim 1 or 2, wherein the skin cancer is non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or adenocarcinoma. **Claim 7** The use according to claim 3, wherein the HAL variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by the HAL is a genomic nucleic acid molecule having a nucleotide sequence comprising adenine or its complement at a position corresponding to position 11,352 as set forth in SEQ ID NO: 2 or guanine or its complement at a position corresponding to position 14,441 as set forth in SEQ ID NO:

3.

8. The use according to claim 3 or 7, wherein the skin cancer is non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or adenocarcinoma.

9. The use according to claim 4 or 5, wherein the HAL mutant nucleic acid molecule encoding a gain-of-function polypeptide predicted by the HAL has a nucleotide sequence containing adenine or its complement at a position corresponding to position 11,352 described in SEQ ID NO: 2 or guanine or its complement at a position corresponding to position 14,441 described in SEQ ID NO: 3, and is a genomic nucleic acid molecule.

10. The use according to claim 4 or 5, wherein the skin cancer is non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or adenocarcinoma.

11. A pharmaceutical composition for treating skin cancer in a subject who is heterozygous or homozygous for a HAL mutant nucleic acid molecule encoding a gain-of-function polypeptide predicted by histidinammonia-lyase (HAL), the pharmaceutical composition comprising, as an active ingredient, a therapeutic agent for treating or preventing skin cancer.

12. A pharmaceutical composition for treating skin cancer in a subject, wherein the subject is a) a reference for a histidinammonia-lyase (HAL) genomic nucleic acid molecule or HAL mRNA molecule, or b) heterozygous for a HAL mutant genomic nucleic acid molecule or HAL mutant mRNA molecule encoding a gain-of-function polypeptide predicted by HAL, and the pharmaceutical composition comprises, as an active ingredient, a HAL agonist.

13. The pharmaceutical composition according to claim 11 or 12, wherein the skin cancer is non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or adenocarcinoma.

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