Patent Application
20240191304
This application includes a Sequence Listing submitted electronically as a text file named 18530009802SEQ, created on Apr. 20, 2022, with a size of 368 kilobytes. The Sequence Listing is incorporated herein by reference.
The present disclosure relates generally to the treatment of subjects having malignant mesothelioma, methods of identifying subjects having an increased risk of developing malignant mesothelioma, methods of detecting LRRK2 variant nucleic acid molecules, and LRRK2 variant nucleic acid molecules.
Malignant mesothelioma (MM) is an aggressive cancer generally associated with asbestos exposure. Individuals with MM, especially the pleural form of this disease, have a poor prognosis due to the dearth of opportunities for early surgical intervention and relatively ineffective chemotherapies. MM causes about 3,200 deaths annually in the U.S., and the incidence of MM is expected to increase 5-10% per year over the next two decades in Europe.
In 2011, two germline mutations of the BAP1 gene were reported in two families, dubbed L and W, with high incidence of MM and other cancers (Testa et al., Nat. Genet., 2011, 43, 1022-1025). Immunohistochemical analysis of MMs from these families revealed a lack of BAP1 nuclear expression and only weak cytoplasmic BAP1 staining. Interestingly, in the L family, two family members had uveal melanoma (UM), one of whom later developed MM.
Notably, inactivating somatic mutations of BAP1 were previously uncovered in about 85% of metastasizing UMs, and one of their patients had a germline mutation in BAPI, suggesting the existence of a tumor susceptibility allele in this individual (Harbour et al., Science, 2010, 330, 1410-1413). Simultaneously, inactivating germline BAP1 mutations were described in two -2-families with multiple benign melanocytic tumors; furthermore, some of the affected individuals developed cutaneous melanomas or an occasional UM, and one family member was later diagnosed with peritoneal MM (Wiesner et al., Nat. Genet., 2011, 43, 1018-10121). Since then, there have been many other reports documenting germline BAP1 mutations in families with MM and various other cancers. Collectively, these findings suggested the existence of a single BAP1 tumor predisposition syndrome (BAP1-TPDS) in which affected families are predisposed primarily to MM, UM, cutaneous melanoma, benign melanocytic tumors, clear cell renal cell carcinoma, and basal cell carcinoma. In a comprehensive assessment of the clinical phenotype of BAP1 variant-carrying families worldwide, the classically described core tumor spectrum for the BAP1-TPDS has been expanded to include meningioma and cholangiocarcinoma, based on the associated much higher incidence of these two rare neoplasms than in the general population, as well as on molecular evidence from tumors of these carriers. Other unconfirmed tumors in the BAP1-TPDS are thought to include breast cancer, non-small cell lung adenocarcinoma, and neuroendocrine carcinoma. Biallelic inactivation of BAP1 has been documented in multiple tumors from these high-risk BAP1 families, implying that BAP1 acts as a classical tumor suppressor gene. Consistent with these riveting genetic discoveries, BAP1 has also been found to exhibit tumor suppressor activity both in cell-based transfection assays and in vivo studies with genetically engineered mice. Furthermore, tumor suppression requires both nuclear localization and BAP1 deubiquitinase activity.
Despite the identification of germline mutations in BAP1 as predisposing to MM and other cancers, there are a number of recent reports indicating that this gene may not account for all examples of familial MM or for all high-risk cancer families with at least one MM. For example, in one study, an Italian family with 8 confirmed cases of MM did not exhibit a predisposing BAP1 mutation. In another study, the entire BAP1 gene was sequenced in blood samples from 150 MM cases with a history of asbestos exposure and a past personal or family history of one or more of the cancers reported in BAP1-TPDS families. These cases all had high “cancer signal” scores, based on an objective scoring system designed to identify MM individuals with a strong family history of cancers including at least some known to be part of the BAP1-TPDS, particularly MM, cutaneous and uveal melanoma, and renal cell carcinoma. Sequencing of BAP1 in these 150 MM cases uncovered 9 patients (6%) with germline mutations affecting the coding sequence of the BAP1 gene.
Current evidence indicates that genetically susceptible individuals are at elevated risk of MM when exposed to asbestos. Due to the presence of asbestos in older buildings and its persistent use in developing countries and in some settings in the U.S., as well as the long latency of MM development, MM will continue to be a health burden for decades. Therefore, understanding how inherited mutations in genes can lead to increased risk for developing MM is highly important. Identifying such high-risk individuals will allow for the implementation of proactive measures, such as biannual ultrasound or MRI imaging, annual physical examinations and serum marker monitoring, and education-related self-awareness among high-risk individuals when presented with disease symptoms.
The present disclosure provides methods of identifying a subject having an increased risk for developing malignant mesothelioma, wherein the method comprises: determining or having determined the presence or absence of a Leucine-Rich Repeat Kinase 2 (LRRK2) variant nucleic acid molecule encoding an LRRK2 polypeptide in a biological sample obtained from the subject; wherein: when the subject does not have an LRRK2 variant nucleic acid molecule, then the subject does not have an increased risk for developing malignant mesothelioma; and when the subject is heterozygous or homozygous for an LRRK2 variant nucleic acid molecule, then the subject has an increased risk for developing malignant mesothelioma.
The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits malignant mesothelioma, wherein the subject has malignant mesothelioma, the method comprising the steps of: determining whether the subject has an LRRK2 variant nucleic acid molecule encoding an LRRK2 polypeptide by: obtaining or having obtained a biological sample from the subject; and performing or having performed an assay on the biological sample to determine if the subject has a genotype comprising the LRRK2 variant nucleic acid molecule; and when the subject does not have an LRRK2 variant nucleic acid molecule, then administering or continuing to administer to the subject the therapeutic agent that treats or inhibits malignant mesothelioma in a standard dosage amount; and when the subject is heterozygous or homozygous for an LRRK2 variant nucleic acid molecule, then administering or continuing to administer to the subject the therapeutic agent that treats or inhibits malignant mesothelioma in an amount that is the same as or greater than a standard dosage amount; wherein the presence of a genotype having the LRRK2 variant nucleic acid molecule encoding the LRRK2 polypeptide indicates the subject has an increased risk of developing malignant mesothelioma.
The present disclosure also provides methods detecting an LRRK2 variant nucleic acid molecule in a subject comprising assaying a sample obtained from the subject to determine whether a nucleic acid molecule in the sample comprises a nucleotide sequence comprising a -4-deletion of the nucleotides AAAGGTAAGG (SEQ ID NO:3) located at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof.
The present disclosure also provides isolated alteration-specific probes or alteration-specific primers comprising at least about 15 nucleotides, wherein the alteration-specific probes or alteration-specific primers comprise a nucleotide sequence which is complementary to a portion of a nucleotide sequence encoding a human LRRK2 polypeptide, wherein the portion comprises positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof, or positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof.
The present disclosure also provides isolated nucleic acid molecules comprising a nucleotide sequence encoding a human LRRK2 polypeptide, wherein the nucleotide sequence comprises a deletion of the nucleotides AAAGGTAAGG (SEQ ID NO:3) located at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof.
The present disclosure also provides therapeutic agents that treat or inhibit malignant mesothelioma for use in the treatment of malignant mesothelioma in a subject having a genomic nucleic acid molecule having a nucleotide sequence encoding an LRRK2 polypeptide, wherein the nucleotide sequence comprises a deletion of the nucleotides AAAGGTAAGG (SEQ ID NO:3) located at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof.
The present disclosure also provides therapeutic agents that treat or inhibit malignant mesothelioma for use in the preparation of a medicament for treating malignant mesothelioma in a subject having a genomic nucleic acid molecule having a nucleotide sequence encoding an LRRK2 polypeptide, wherein the nucleotide sequence comprises a deletion of the nucleotides AAAGGTAAGG (SEQ ID NO:3) located at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the present disclosure.
Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.
Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-expressed basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term “about” means that the recited numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments.
Where a numerical value is used, unless indicated otherwise by the context, the term “about” means the numerical value can vary by +10% and remain within the scope of the disclosed embodiments.
As used herein, the term “comprising” may be replaced with “consisting” or “consisting essentially of” in particular embodiments as desired.
As used herein, the term “isolated”, in regard to a nucleic acid molecule or a polypeptide, means that the nucleic acid molecule or polypeptide is in a condition other than its native environment, such as apart from blood and/or animal tissue. 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.
As used herein, the terms “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “polynucleotide”, or “oligonucleotide” can comprise a polymeric form of nucleotides of any length, can comprise DNA and/or RNA, and can be single-stranded, double-stranded, or multiple stranded. One strand of a nucleic acid also refers to its complement.
As used herein, the term “subject” includes any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horse, cow, pig), companion animals (such as, for example, dog, cat), laboratory animals (such as, for example, mouse, rat, rabbits), and non-human primates (such as, for example, 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.
A variant in the LRRK2 gene associated with an increased risk of developing malignant mesothelioma in humans has been identified in accordance with the present disclosure. For example, a genetic alteration that deletes nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions 97,182 to 97,191 in the human LRRK2 reference (see, SEQ ID NO:1) (hereinafter termed “LRRK2 AAAAGGTAAGG”) has been observed to indicate that the human having such an alteration may have an increased risk of developing malignant mesothelioma. It is believed that no variants of the LRRK2 gene or protein have any known association with a malignant mesothelioma. The genetic analyses described herein surprisingly indicate that the LRRK2 gene and, in particular, a variant in the LRRK2 gene, associates with an increased risk of developing malignant mesothelioma. Therefore, humans that have an LRRK2 variant nucleic acid molecule that associates with an increased risk of developing malignant mesothelioma, may be treated -8-such that the malignant mesothelioma is prevented, the symptoms thereof are reduced, and/or development of symptoms is repressed. Accordingly, the present disclosure provides methods of leveraging the identification of such LRRK2 variants in subjects to identify or stratify risk in such subjects of developing malignant mesothelioma, or to diagnose subjects as having an increased risk of developing malignant mesothelioma, such that subjects at risk or subjects with active disease may be treated. Additionally, the present disclosure provides isolated LRRK2 variant genomic nucleic acid molecules. Also provided herein are LRRK2 loss-of-function variant nucleic acid molecules discovered to be associated with an increased risk of developing malignant mesothelioma.
For purposes of the present disclosure, any particular human can be categorized as having one of three LRRK2 genotypes: i) LRRK2 reference; ii) heterozygous for an LRRK2 predicted loss-of-function variant; or iii) homozygous for an LRRK2 predicted loss-of-function variant. A human is LRRK2 reference when the human does not have a copy of an LRRK2 predicted loss-of-function variant nucleic acid molecule. A human is heterozygous for an LRRK2 predicted loss-of-function variant when the human has a single copy of an LRRK2 predicted loss-of-function variant nucleic acid molecule. An LRRK2 predicted loss-of-function variant nucleic acid molecule is any LRRK2 nucleic acid molecule (such as, a genomic nucleic acid molecule) encoding an LRRK2 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function.
The LRRK2 predicted loss-of-function variant nucleic acid molecule can be any nucleic acid molecule encoding LRRK2 AAAAGGTAAGG. In some embodiments, the LRRK2 predicted loss-of-function variant nucleic acid molecule encodes LRRK2 AAAAGGTAAGG. A human is homozygous for an LRRK2 predicted loss-of-function variant when the human has two copies of an LRRK2 predicted loss-of-function variant nucleic acid molecule.
For subjects that are genotyped or determined to be heterozygous or homozygous for an LRRK2 predicted loss-of-function variant nucleic acid molecule, such subjects have an increased risk of developing malignant mesothelioma. For subjects that are genotyped or determined to be heterozygous or homozygous for an LRRK2 predicted loss-of-function variant nucleic acid molecule, such subjects can be treated with an agent effective to treat malignant mesothelioma. In some embodiments, subjects who have not yet developed malignant mesothelioma or in which detectable malignant mesothelioma is not yet present, such subjects can be treated with a chemopreventive agent that is potentially less toxic than a chemotherapeutic agent.
The present disclosure provides methods of treating a subject with a therapeutic agent that treats or inhibits malignant mesothelioma, wherein the subject is suffering from malignant mesothelioma, the method comprising the steps of: determining whether the subject has an LRRK2 predicted loss-of-function variant nucleic acid molecule encoding a human LRRK2 polypeptide by: obtaining or having obtained a biological sample from the subject; and performing or having performed an assay on the biological sample to determine if the subject has a genotype comprising the LRRK2 predicted loss-of-function variant nucleic acid molecule; and when the subject is LRRK2 reference, then administering or continuing to administer to the subject the therapeutic agent that treats or inhibits the malignant mesothelioma in a standard dosage amount; and when the subject is heterozygous or homozygous for an LRRK2 predicted loss-of-function variant, then administering or continuing to administer to the subject the therapeutic agent that treats or inhibits the malignant mesothelioma in an amount that is the same as or greater than a standard dosage amount; wherein the presence of a genotype having the LRRK2 predicted loss-of-function variant nucleic acid molecule encoding the human LRRK2 polypeptide indicates the subject has an increased risk of developing the malignant mesothelioma. In some embodiments, the subject is heterozygous for an LRRK2 predicted loss-of-function variant. In some embodiments, the subject is homozygous for an LRRK2 predicted loss-of-function variant.
In some embodiments, the treatment methods further comprise detecting the presence or absence of an LRRK2 predicted loss-of-function variant nucleic acid molecule encoding a human LRRK2 polypeptide in a biological sample from the subject. As used throughout the present disclosure, an “LRRK2 predicted loss-of-function variant nucleic acid molecule” is any LRRK2 nucleic acid molecule (such as, for example, genomic nucleic acid molecule) encoding an LRRK2 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function.
In any of the embodiments described herein, the LRRK2 predicted loss-of-function variant nucleic acid molecule can be any LRRK2 nucleic acid molecule (such as, for example, genomic nucleic acid molecule) encoding an LRRK2 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function. For example, the LRRK2 predicted loss-of-function variant nucleic acid molecule can be any nucleic acid molecule encoding LRRK2 AAAAGGTAAGG. In some embodiments, the LRRK2 predicted loss-of-function variant nucleic acid molecule encodes LRRK2 AAAAGGTAAGG.
The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits malignant mesothelioma, wherein the subject has malignant mesothelioma, the method comprising the steps of: determining whether the subject has a reduced expression of an LRRK2 polypeptide in a tumor cell by: obtaining or having obtained a biological sample from the subject; and performing or having performed an assay on the biological sample to determine if the subject has a reduced expression of an LRRK2 polypeptide; and when the subject does not have a reduced expression of an LRRK2 polypeptide, then administering or continuing to administer to the subject the therapeutic agent that treats or inhibits malignant mesothelioma in a standard dosage amount; and when the subject has a reduced expression of an LRRK2 polypeptide, then administering or continuing to administer to the subject the therapeutic agent that treats or inhibits malignant mesothelioma in an amount that is the same as or greater than a standard dosage amount, and administering a therapeutic agent suitable for treating LRRK2 loss; wherein the reduced expression of the LRRK2 polypeptide indicates the subject has an increased risk of developing malignant mesothelioma.
In any of the embodiments described herein, the LRRK2 predicted loss-of-function variant nucleic acid molecule can be any LRRK2 nucleic acid molecule (such as, for example, genomic nucleic acid molecule) encoding an LRRK2 polypeptide whose expression in a tumor cell is partially reduced or eliminated. For example, it has been observed that tumors from more than 50% of mesothelioma patients may have complete or partial loss of LRRK2 protein expression, based on immunoblot analysis.
Detecting the presence or absence of an LRRK2 predicted loss-of-function variant nucleic acid molecule in a biological sample from a subject and/or determining whether a subject has an LRRK2 predicted loss-of-function variant nucleic acid molecule can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the nucleic acid molecule can be present within a cell obtained from the subject.
In any of the embodiments described herein, the LRRK2 predicted loss-of-function polypeptide can be any LRRK2 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function. In any of the embodiments described herein, the LRRK2 predicted loss-of-function polypeptide can be any of the LRRK2 polypeptides described herein including, for example, LRRK2 AAAAGGTAAGG. In some embodiments, the LRRK2 predicted loss-of-function polypeptide is LRRK2 AAAAGGTAAGG.
The present present disclosure also provides methods of identifying subjects having reduced LRRK2 polypeptide expression or no LRRK2 polypeptide expression in a malignant mesothelioma tumor cell. Such methods include, but are not limited to, immunohistochemistry.
Such subjects can be treated with targeted therapies relevant to LRRK2 loss, as described herein. Symptoms of malignant mesothelioma include, but are not limited to, chest pain, painful coughing, shortness of breath, unusual lumps of tissue under the skin on your chest, unexplained weight loss, fatigue, loss of appetite, night sweats, bloating or nausea, hoarseness, reduced chest expansion, abdominal pain, abdominal swelling, abdominal fluid buildup (ascites), and constipation.
Examples of therapeutic agents that treat or inhibit malignant mesothelioma include, but are not limited to, standard chemotherapy agents, such as platinum-based chemotherapeutics (e.g., cisplatin or carboplatin) combined with pemetrexed; immune checkpoint inhibitors (e.g., nivolumab and ipilimumab); autophagy inhibitors: (e.g., chloroquine and hydroxychloroquine); PARP inhibitors (e.g., olaparib, rucaparib, niraparib, and talazoparib); histone acetylation-targeted drugs (e.g., valproic acid, phenylbutyrate, vorinostat, belinostat, romidepsin, nicotinamide, and GSK525762); and DNA methyltransferase (DNMT) inhibitors: (e.g., azacitideine, decitabine, and hydralazine).
In some embodiments, the dose of the therapeutic agents that treat or inhibit malignant mesothelioma can be increased by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, or by about 90% for subjects that are heterozygous or homozygous for an LRRK2 predicted loss-of-function variant (i.e., a greater amount than the standard dosage amount) compared to subjects that are LRRK2 reference (who may receive a standard dosage amount). In some embodiments, the dose of the therapeutic agents that treat or inhibit malignant mesothelioma can be increased by about 10%, by about 20%, by about 30%, by about 40%, or by about 50%. In addition, the dose of therapeutic agents that treat or inhibit malignant mesothelioma in subjects that are heterozygous or homozygous for an LRRK2 predicted loss-of-function variant can be administered more frequently compared to subjects that are LRRK2 reference.
In some embodiments, the dose of the therapeutic agents that treat or inhibit malignant mesothelioma can be increased by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, or by about 90% for subjects that are homozygous for an LRRK2 predicted loss-of-function variant compared to subjects that are heterozygous for an LRRK2 predicted loss-of-function variant. In some embodiments, the dose of the therapeutic agents that treat or inhibit malignant mesothelioma can be increased by about 10%, by about 20%, by about 30%, by about 40%, or by about 50%. In addition, the dose of therapeutic agents that treat or inhibit the malignant mesothelioma in subjects that are homozygous for an LRRK2 predicted loss-of-function variant can be administered more frequently compared to subjects that are heterozygous for an LRRK2 predicted loss-of-function variant.
Administration of therapeutic agents that treat or inhibit malignant mesothelioma can be repeated, for example, after one day, two days, three days, five days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, eight weeks, two months, or three months. The repeated administration can be at the same dose or at a different dose. The administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. For example, according to certain dosage regimens a subject can receive therapy for a prolonged period of time such as, for example, 6 months, 1 year, or more.
Administration of therapeutic agents that treat or inhibit malignant mesothelioma can occur by any suitable route including, but not limited to, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. The term “pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.
The terms “treat”, “treating”, and “treatment” and “prevent”, “preventing”, and “prevention” as used herein, refer to eliciting the desired biological response, such as therapeutic and prophylactic effect, respectively. In some embodiments, therapeutic effect comprises one or more of a decrease/reduction in malignant mesothelioma, a decrease/reduction in the severity of malignant mesothelioma (such as, for example, a reduction or inhibition of development of malignant mesothelioma), a decrease/reduction in symptoms and malignant mesothelioma-related effects, delaying the onset of symptoms and malignant mesothelioma-related effects, reducing the severity of symptoms of malignant mesothelioma-related effects, reducing the severity of an acute episode, reducing the number of symptoms and malignant mesothelioma-related effects, reducing the latency of symptoms and malignant mesothelioma-related effects, an amelioration of symptoms and malignant mesothelioma-related effects, reducing secondary symptoms, reducing secondary infections, preventing relapse to malignant mesothelioma, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, increasing time to sustained progression, expediting remission, inducing remission, augmenting remission, speeding recovery, or increasing efficacy of or decreasing resistance to alternative therapeutics, and/or an increased survival time of the affected host animal, following administration of the agent or composition comprising the agent. A prophylactic effect may comprise a complete or partial avoidance/inhibition or a delay of malignant mesothelioma development/progression (such as, for example, a complete or partial avoidance/inhibition or a delay), and an increased survival time of the affected host animal, following administration of therapeutic protocol. Treatment of malignant mesothelioma encompasses the treatment of subjects already diagnosed as having any form of malignant mesothelioma at any clinical stage or manifestation, the delay of the onset or evolution or aggravation or deterioration of the symptoms or signs of malignant mesothelioma, and/or preventing and/or reducing the severity of malignant mesothelioma.
The present disclosure also provides methods of identifying a subject having an increased risk for developing malignant mesothelioma, wherein the method comprises: determining or having determined in a biological sample obtained from the subject the presence or absence of an LRRK2 predicted loss-of-function variant nucleic acid molecule (such as a genomic nucleic acid molecule) encoding a human LRRK2 polypeptide; wherein: i) when the subject lacks an LRRK2 predicted loss-of-function variant nucleic acid molecule (i.e., the subject is genotypically categorized as an LRRK2 reference), then the subject does not have an increased risk for developing malignant mesothelioma; and ii) when the subject has an LRRK2 predicted loss-of-function variant nucleic acid molecule (i.e., the subject is heterozygous or homozygous for an LRRK2 predicted loss-of-function variant), then the subject has an increased risk for developing malignant mesothelioma.
In any of the embodiments described herein, the LRRK2 predicted loss-of-function variant nucleic acid molecule can be any LRRK2 nucleic acid molecule (such as, for example, genomic nucleic acid molecule) encoding an LRRK2 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function. For example, the LRRK2 predicted loss-of-function variant nucleic acid molecule can be any nucleic acid molecule encoding LRRK2 AAAAGGTAAGG. In some embodiments, the LRRK2 predicted loss-of-function variant nucleic acid molecule encodes LRRK2 AAAAGGTAAGG.
Determining whether a subject has an LRRK2 predicted loss-of-function variant nucleic acid molecule in a biological sample from a subject and/or determining whether a subject has an LRRK2 predicted loss-of-function variant nucleic acid molecule can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the nucleic acid molecule can be present within a cell obtained from the subject.
In some embodiments, when a subject is identified as having an increased risk of developing malignant mesothelioma, the subject is treated with a therapeutic agent that treats or inhibits malignant mesothelioma, as described herein. In some embodiments, when the subject is heterozygous or homozygous for an LRRK2 predicted loss-of-function variant, the subject is administered the therapeutic agent that treats or inhibits malignant mesothelioma in a dosage amount that is the same as or greater than the standard dosage amount. In some embodiments, when the subject is homozygous for an LRRK2 predicted loss-of-function variant, the subject is administered the therapeutic agent that treats or inhibits malignant mesothelioma in a dosage amount that is the same as or greater than the dosage amount administered to a subject that is heterozygous for an LRRK2 predicted loss-of-function variant. In some embodiments, the subject is heterozygous for an LRRK2 predicted loss-of-function variant. In some embodiments, the subject is homozygous for an LRRK2 predicted loss-of-function variant.
The present disclosure also provides methods of detecting the presence or absence of an LRRK2 predicted loss-of-function variant genomic nucleic acid molecule in a biological sample from a subject. It is understood that gene sequences within a population and mRNA molecules encoded by such genes can vary due to polymorphisms such as single-nucleotide polymorphisms. The sequences provided herein for the LRRK2 variant genomic nucleic acid molecule, are only exemplary sequences. Other sequences for the LRRK2 variant genomic nucleic acid molecule are also possible.
The biological sample can be derived from any cell, tissue, or biological fluid from the subject. The sample may comprise any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of bodily fluid, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascitic fluid, cystic fluid, or urine. In some cases, the sample comprises a buccal swab. The sample used in the methods disclosed herein will vary based on the assay format, nature of the detection method, and the tissues, cells, or extracts that are used as the sample.
In some embodiments, detecting an LRRK2 predicted loss-of-function variant nucleic acid molecule in a subject comprises assaying or genotyping a biological sample obtained from the subject to determine whether an LRRK2 genomic nucleic acid molecule in the biological sample, comprises one or more variations that cause a loss-of-function (partial or complete) or are predicted to cause a loss-of-function (partial or complete).
In some embodiments, the methods of detecting the presence or absence of an LRRK2 predicted loss-of-function variant nucleic acid molecule (such as, for example, a genomic nucleic acid molecule) in a subject, comprise: performing an assay on a biological sample obtained from the subject, which assay determines whether a nucleic acid molecule in the biological sample comprises a particular nucleotide sequence. In some embodiments, the nucleotide sequence comprises a deletion of the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1 (for genomic nucleic acid molecules). In some embodiments, the nucleotide sequence comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2 (for genomic nucleic acid molecules).
In some embodiments, the biological sample comprises a cell or cell lysate. Such methods can further comprise, for example, obtaining a biological sample from the subject comprising an LRRK2 genomic nucleic acid molecule. Such assays can comprise, for example determining the identity of these positions of the particular LRRK2 nucleic acid molecule. In some embodiments, the method is an in vitro method.
In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the LRRK2 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises one or more variations that cause a loss-of-function (partial or complete) or are predicted to cause a loss-of-function (partial or complete).
In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion o the nucleotide sequence of the LRRK2 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises nucleotides at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof. When the sequenced portion of the LRRK2 nucleic acid molecule in the biological sample comprises a deletion of the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, then the LRRK2 nucleic acid molecule in the biological sample is an LRRK2 predicted loss-of-function variant nucleic acid molecule.
In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the LRRK2 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof. When the sequenced portion of the LRRK2 nucleic acid molecule in the biological sample comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, then the LRRK2 nucleic acid molecule in the biological sample is an LRRK2 predicted loss-of-function variant nucleic acid molecule.
In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) contacting the biological sample with a primer hybridizing to a portion of the nucleotide sequence of the LRRK2 genomic nucleic acid molecule that is proximate to positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1; b) extending the primer at least through the position of the nucleotide sequence of the LRRK2 genomic nucleic acid molecule corresponding to position 97,182 to 97,191 according to SEQ ID NO:1; and c) determining whether the extension product of the primer comprises a deletion of nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1.
In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) contacting the biological sample with a primer hybridizing to a portion of the nucleotide sequence of the LRRK2 genomic nucleic acid molecule that is proximate to positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2; b) extending the primer at least through the position of the nucleotide sequence of the LRRK2 genomic nucleic acid molecule corresponding to position 97,181 to 97,190 according to SEQ ID NO:2; and c) determining whether the extension product of the primer comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2.
In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only an LRRK2 genomic nucleic acid molecule is analyzed.
In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of the nucleic acid molecule that encodes the human LRRK2 polypeptide, wherein the amplified portion comprises the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof, b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule comprising the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof; and d) detecting the detectable label.
In some embodiments, the determining step, detecting step, or genotyping assay comprises: a) amplifying at least a portion of the nucleic acid molecule that encodes the human LRRK2 polypeptide, wherein the amplified portion comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule comprising the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof; and d) detecting the detectable label.
In some embodiments, the determining step, detecting step, or genotyping assay comprises: contacting the nucleic acid molecule in the biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof; and detecting the detectable label.
In some embodiments, the determining step, detecting step, or genotyping assay comprises: contacting the nucleic acid molecule in the biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe comprises a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising: the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof; and detecting the detectable label.
In some embodiments, the nucleic acid molecule is present within a cell obtained from the subject.
The LRRK2 predicted loss-of-function variant nucleic acid molecule can be any LRRK2 nucleic acid molecule (such as, for example, genomic nucleic acid molecule) encoding an LRRK2 polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function. For example, the LRRK2 predicted loss-of-function variant nucleic acid molecule can be any nucleic acid molecule encoding LRRK2 AAAAGGTAAGG. In some embodiments, the LRRK2 predicted loss-of-function variant nucleic acid molecule encodes LRRK2 AAAAGGTAAGG.
In some embodiments, the assay comprises contacting the biological sample with a primer or probe, such as an alteration-specific primer or alteration-specific probe, that specifically hybridizes to an LRRK2 variant genomic sequence and not the corresponding LRRK2 reference sequence under stringent conditions, and determining whether hybridization has occurred.
In some embodiments, to determine whether an LRRK2 nucleic acid molecule (genomic nucleic acid molecule), or complement thereof, within a biological sample comprises a nucleotide sequence comprising the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1 (genomic nucleic acid molecule), the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from the 5′ flanking sequence adjacent to the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, and a second primer derived from the 3′ flanking sequence adjacent to the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1 to produce an amplicon that is indicative of the presence of the deltion of the nucleotides AAAGGTAAGG (SEQ ID NO: 3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO: 1. In some embodiments, the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. This distance can range from one nucleotide base pair up to the limits of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair flanks a region including positions comprising the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of positions comprising the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1.
In some embodiments, to determine whether an LRRK2 nucleic acid molecule (genomic nucleic acid molecule), or complement thereof, within a biological sample comprises a nucleotide sequence comprising the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2 (genomic nucleic acid molecule), the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from the 5′ flanking sequence adjacent to the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, and a second primer derived from the 3′ flanking sequence adjacent to the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2 to produce an amplicon that is indicative of the presence the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2. In some embodiments, the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. This distance can range from one nucleotide base pair up to the limits of the amplification reaction, or about twenty thousand nucleotide base pairs. Optionally, the primer pair flanks a region including positions comprising the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of positions comprising the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2.
Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, including using labeled primers or probes directed against purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, a target nucleic acid molecule may be amplified prior to or simultaneous 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).
Appropriate stringency conditions which promote DNA hybridization, for example, 6X sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2×SSC at 50° C., are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection will be those in which the salt concentration is less than about 1.5 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (such as, for example, 10 to 50 nucleotides) and at least about 60° C. for longer probes (such as, for example, greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
The present present disclosure also provides methods of detecting LRRK2 polypeptide expression. Such methods include, but are not limited to, immunohistochemistry.
The present disclosure also provides isolated nucleic acid molecules that hybridize to LRRK2 variant genomic nucleic acid molecules (such as any of the genomic variant nucleic acid molecules disclosed herein. In some embodiments, the isolated nucleic acid molecules hybridize to a portion of the LRRK2 nucleic acid molecule that includes positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1. In some embodiments, the isolated nucleic acid molecules hybridize to a portion of the LRRK2 nucleic acid molecule that includes positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2.
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, 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 molecules comprise or consist of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 12 to about 30, from about 12 to about 28, from about 12 to about 24, from about 15 to about 30, from about 15 to about 25, from about 18 to about 30, from about 18 to about 25, from about 18 to about 24, or from about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 18 to about 30 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consist of at least about 15 nucleotides to at least about 35 nucleotides.
In some embodiments, such isolated nucleic acid molecules hybridize to LRRK2 variant nucleic acid molecules (such as genomic nucleic acid molecules) under stringent conditions.
Such nucleic acid molecules can be used, for example, as probes, primers, alteration-specific probes, or alteration-specific primers as described or exemplified herein, and include, without limitation primers, probes, antisense RNAs, shRNAs, and siRNAs, each of which is described in more detail elsewhere herein, and can be used in any of the methods described herein.
In some embodiments, the isolated alteration-specific probes or alteration-specific primers comprise at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence which is complementary to a portion of a nucleotide sequence encoding a human LRRK2 polypeptide, wherein the portion comprises a position corresponding to position 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof.
In some embodiments, the isolated alteration-specific probes or alteration-specific primers comprise at least about 15 nucleotides, wherein the alteration-specific probe or alteration-specific primer comprises a nucleotide sequence which is complementary to a portion of a nucleotide sequence encoding a human LRRK2 polypeptide, wherein the portion comprises a position corresponding to position 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof.
In some embodiments, the alteration-specific probes and alteration-specific primers comprise DNA. In some embodiments, the alteration-specific probes and alteration-specific primers comprise RNA.
In some embodiments, the probes and primers described herein (including alteration-specific probes and alteration-specific primers) have a nucleotide sequence that specifically hybridizes to any of the nucleic acid molecules disclosed herein, or the complement thereof. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.
The present disclosure also provides pairs of primers comprising any of the primers described above. If one of the primers' 3′-ends hybridizes anywhere within AAAGGTAAGG sequence (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1 (rather than the nucleotides AAAATCAATT (SEQ ID NO:4)) in a particular LRRK2 nucleic acid molecule, then the presence of the amplified fragment would indicate the presence of an LRRK2 reference genomic nucleic acid molecule. Conversely, if one of the primers' 3′-ends hybridizes to the nucleotides anywhere within AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2 (rather than nucleotides AAAGGTAAGG sequence (SEQ ID NO:3)) in a particular LRRK2 nucleic acid molecule, then the presence of the amplified fragment would indicate the presence of the LRRK2 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to any of the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1 can be at the 3′ end of the primer. In some embodiments, the nucleotide of the primer complementary to any of the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2 can be at the 3′ end of the primer.
In the context of the disclosure “specifically hybridizes” means that the probe or primer (such as, for example, the alteration-specific probe or alteration-specific primer) does not hybridize to a nucleic acid sequence encoding an LRRK2 reference genomic nucleic acid molecule.
In some embodiments, the probes (such as, for example, an alteration-specific probe) comprise a label. In some embodiments, the label is a fluorescent label, a radiolabel, or biotin.
The present disclosure also provides supports comprising a substrate to which any one or more of the probes disclosed herein is attached. Solid supports are solid-state substrates or supports with which molecules, such as any of the probes disclosed herein, can be associated. 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 multiple different probes have been coupled in an array, grid, or other organized pattern. A form for a solid-state substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a multiwell glass slide can be employed that normally contains one array per well.
The present disclosure also provides molecular complexes comprising or consisting of any of the LRRK2 nucleic acid molecules (genomic nucleic acid molecules), or complement thereof, described herein and any of the alteration-specific primers or alteration-specific probes described herein. In some embodiments, the LRRK2 nucleic acid molecules (genomic nucleic acid molecules), or complement thereof, in the molecular complexes are single-stranded. In some embodiments, the LRRK2 nucleic acid molecule is any of the genomic nucleic acid molecules described herein. In some embodiments, the molecular complex comprises or consists of any of the LRRK2 nucleic acid molecules (genomic nucleic acid molecules), or complement thereof, described herein and any of the alteration-specific primers described herein. In some embodiments, the molecular complex comprises or consists of any of the LRRK2 nucleic acid molecules (genomic nucleic acid molecules), or complement thereof, described herein and any of the alteration-specific probes described herein.
In some embodiments, the molecular complex comprises or consists of an alteration-specific primer or an alteration-specific probe hybridized to a genomic nucleic acid molecule comprising a nucleotide sequence encoding an LRRK2 polypeptide, wherein the alteration-specific primer or the alteration-specific probe is hybridized to the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof, or the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof. In some embodiments, the molecular complex comprises or consists of a genomic nucleic acid molecule that comprises SEQ ID NO:1 or SEQ ID NO:2.
In some embodiments, the molecular complex comprises an alteration-specific probe or an alteration-specific primer comprising a label. In some embodiments, the label is a fluorescent label, a radiolabel, or biotin. In some embodiments, the molecular complex further comprises a non-human polymerase.
The nucleotide sequence of an LRRK2 reference genomic nucleic acid molecule is set forth in SEQ ID NO:1 (GRCh38/hg38 chrl2:40,224,997-40,369,285 ENST00000298910.12). Referring to SEQ ID NO:1, positions 97,182 to 97,191 is the AAAGGTAAGG (SEQ ID NO:3).
A variant genomic nucleic acid molecule of LRRK2 exists (del(AAAGGTAAGG) at positions 97,182-97,191 of SEQ ID NO:1 corresponding to GRCh38/hg38 chrl2:40,322,178-40,322,187 deleted), wherein the AAAGGTAAGG (SEQ ID NO:3) at positions 97,182 to 97,191 deleted. The nucleotide sequence of this LRRK2 variant genomic nucleic acid molecule is set forth in SEQ ID NO:2.
The present disclosure also provides isolated genomic nucleic acid molecules comprising or consisting of a nucleotide sequence encoding a human LRRK2 polypeptide. In some embodiments, the nucleotide sequence of the genomic nucleic acid molecule comprises the deletion of nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof. In some embodiments, the nucleotide sequence of the genomic nucleic acid molecule comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof.
In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:1, and lacks nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 90% sequence identity to SEQ ID NO: 1, and lacks nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 92% sequence identity to SEQ ID NO: 1, and lacks nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 94% sequence identity to SEQ ID NO:1, and lacks nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 96% sequence identity to SEQ ID NO:1, and lacks nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 98% sequence identity to SEQ ID NO:1, and lacks nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof.
In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:2, and comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 90% sequence identity to SEQ ID NO:2, and comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 92% sequence identity to SEQ ID NO:2, and comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 94% sequence identity to SEQ ID NO:2, and comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 96% sequence identity to SEQ ID NO:2, and comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof. In some embodiments, the isolated genomic nucleic acid molecules comprise or consist of a nucleotide sequence that has at least about 98% sequence identity to SEQ ID NO:2, and comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof.
In some embodiments, the isolated genomic nucleic acid molecules comprise SEQ ID NO:2. In some embodiments, the isolated genomic nucleic acid molecules consist of SEQ ID NO:2.
The genomic nucleic acid molecules can be from any organism. For example, the genomic nucleic acid molecules can be human or an ortholog from another organism, such as a non-human mammal, a rodent, a mouse, or a rat. It is understood that gene sequences within a population can vary due to polymorphisms such as single-nucleotide polymorphisms. The examples provided herein are only exemplary sequences. Other sequences are also possible.
The isolated nucleic acid molecules disclosed herein can comprise RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence, such as in a vector, or a heterologous label. For example, the isolated nucleic acid molecules disclosed herein can be within a vector or as an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to a heterologous label. The label can be directly detectable (such as, for example, fluorophore) or indirectly detectable (such as, for example, hapten, enzyme, or fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radiolabels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal. The term “label” can also refer to a “tag” or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal. For example, biotin can be used as a tag along with an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and examined using a calorimetric substrate (such as, for example, tetramethylbenzidine (TMB)) or a 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, an epitope tag, or the Fc portion of immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorogenic and chemiluminescent substrates and other labels.
The disclosed nucleic acid molecules can comprise, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include a nucleotide that contains a modified base, sugar, or phosphate group, or that incorporates a non-natural moiety in its structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophor-labeled nucleotides.
The nucleic acid molecules disclosed herein can also comprise one or more nucleotide analogs or substitutions. A nucleotide analog is a nucleotide which contains a modification to either the base, sugar, or phosphate moieties. 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 different purine or pyrimidine bases such as, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. Modified bases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 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,-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (such as, for example, 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.
Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of the ribose and deoxy ribose 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; 0-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-10alkyl or C2-10alkenyl, and C2-10alkynyl. Exemplary 2′ sugar modifications also include, but are not limited to, —O[(CH2)nO]mCH3, —O(CH2)nOCH3, —O(CH2)nNH2, —O(CH2)nCH3, —O(CH2)n—ONH2, and —O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other modifications at the 2′ position include, but are not limited to, C1-10alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Modified sugars can also include those that contain 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.
Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3′-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. These phosphate or modified phosphate linkage between two nucleotides can be through a 3′-5′ linkage or a 2′-5′ linkage, and the linkage can contain inverted 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).
The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5′ end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3′ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The amino acid sequence follows the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.
The present disclosure also provides vectors comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vectors comprise any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vectors can be viral or nonviral vectors capable of transporting a nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid (such as, for example, a circular double-stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector, wherein 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.
Percent identity (or percent complementarity) between particular stretches of nucleotide sequences within nucleic acid molecules or amino acid sequences within polypeptides can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Herein, if reference is made to percent sequence identity, the higher percentages of sequence identity are preferred over the lower ones.
The present disclosure also provides compositions comprising any one or more of the isolated nucleic acid molecules, such as genomic nucleic acid molecules disclosed herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the compositions comprise a carrier and/or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochleates, and lipid microtubules. A carrier may comprise a buffered salt solution such as PBS, HBSS, etc.
As used herein, the phrase “corresponding to” or grammatical variations thereof when used in the context of the numbering of a particular nucleotide or nucleotide sequence or position refers to the numbering of a specified reference sequence when the particular nucleotide or nucleotide sequence is compared to a reference sequence (such as, for example, SEQ ID NO:1).
In other words, the residue (such as, for example, nucleotide or amino acid) number or residue (such as, for example, nucleotide or amino acid) position of a particular polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the particular nucleotide or nucleotide sequence is made with respect to the reference sequence to which it has been aligned.
For example, a nucleic acid molecule comprising a nucleotide sequence encoding a human LRRK2 polypeptide, wherein the nucleotide sequence comprises the deletion of the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1 means that if the nucleotide sequence of the LRRK2 genomic nucleic acid molecule is aligned to the sequence of SEQ ID NO:1, the LRRK2 sequence has the deletion of the nucleotides AAAGGTAAGG (SEQ ID NO:3) residue at the position that corresponds to position 97,182 to 97,191 of SEQ ID NO:1.
As described herein, a position within an LRRK2 genomic nucleic acid molecule that corresponds to position 97,182 to 97,191 according to SEQ ID NO:1, for example, can be identified by performing a sequence alignment between the nucleotide sequence of a particular LRRK2 nucleic acid molecule and the nucleotide sequence of SEQ ID NO:1. A variety of computational algorithms exist that can be used for performing a sequence alignment to identify a nucleotide position that corresponds to, for example, position 97,182 to 97,191 in SEQ ID NO:1. For example, 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) sequence alignments may be performed. However, sequences can also be aligned manually.
The present disclosure also provides therapeutic agents that treat or inhibit malignant mesothelioma for use in the treatment of malignant mesothelioma (or for use in the preparation of a medicament for treating malignant mesothelioma) in a subject, wherein the subject has any of the genomic nucleic acid molecules encoding an LRRK2 polypeptide described herein. The therapeutic agents that treat or inhibit malignant mesothelioma can be any of the therapeutic agents that treat or inhibit malignant mesothelioma described herein.
In some embodiments, the subject has a genomic nucleic acid molecule having a nucleotide sequence encoding an LRRK2 polypeptide, wherein the nucleotide sequence comprises a deletion of the nucleotides AAAGGTAAGG (SEQ ID NO:3) at positions corresponding to positions 97,182 to 97,191 according to SEQ ID NO:1, or the complement thereof.
In some embodiments, the subject has a genomic nucleic acid molecule having a nucleotide sequence encoding an LRRK2 polypeptide, wherein the nucleotide sequence comprises the nucleotides AAAATCAATT (SEQ ID NO:4) at positions corresponding to positions 97,181 to 97,190 according to SEQ ID NO:2, or the complement thereof.
All patent documents, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently 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 can be used in combination with any other feature, step, element, embodiment, or aspect unless specifically indicated otherwise. 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.
The following examples are provided to describe the embodiments in greater detail. They are intended to illustrate, not to limit, the claimed embodiments. The following examples provide those of ordinary skill in the art with a disclosure and description of how the compounds, compositions, articles, devices and/or methods described herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (such as, for example, amounts, temperature, etc.), but some errors and deviations may be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
Patients and Samples: Twelve U.S. or Canadian cases of MM were from a series of 141 MM patients with a family history of cancer but with no germline mutation of BAP1. All 12 cases had a known history of asbestos exposure and were identified via one or more of the following: 1) patient health care providers; 2) a patient support organization (Mesothelioma Applied Research Foundation); or 3) independent medical evaluations for medical-legal purposes. The original 141 MM cases were selected based on a personal or family history of one or more of the cancers previously reported in BAP1-TPDS families; however, for the present study of 12 selected cases, the main criterion was a high overall incidence of cancer generally, not necessarily a personal or family history strongly indicative of BAP1-TPDS. Study entry criteria consisted of a pathology report, including immunohistochemical staining confirming the diagnosis of MM, from a CLIA-certified U.S. or Canadian laboratory.
Three Italian cases of MM (2 analyzed by WGS and 1 examined by Sanger sequencing) were from an asbestos-exposed family with multiple cases of pleural MM without inheritance of a predisposing BAP1 mutation. Between 1987 and 2012, 6 women and 2 men developed pleural MM in one generation (generation III). In addition to the 8 confirmed MMs, 2 female family members in generation II had pleural cancers (highly suspected to be MM, but unconfirmed), without radiological evidence of a primary tumor in the lung or elsewhere. The kindred had known exposure to crocidolite asbestos in the domestic setting, as documented by transmission electron microscopy in several family members.
For all of the U.S. and Canadian MM cases, DNA was isolated from blood using standard techniques. For the Italian MM family, DNA for WGS was isolated from peripheral blood in one MM case and from an OCT-embedded sample containing a mixture of MM tumor and normal tissue. A third DNA sample from the Italian family was obtained from a macrodissected tumor of another family member with MM.
Next Generation Sequencing: WGS of genomic DNA isolated from blood was performed using an Illumina HiSeq X Ten platform with paired-end 150-bp reads, with approximately 30-50x coverage. FASTQ files generated from the runs were processed by Novogene, with mapping to the human reference genome (b37) using the Burrows-Wheeler Aligner (BWA). Read alignment BAM files were generated after SAMtools sorting and Picard marking of duplicates. GATK was used to call single nucleotide polymorphisms (SNPs) and small insertions/deletions (small indels) from BAM files. ANNOVAR was used to annotate the variants, which was then scored using the CADD program. Structural variants (SVs) consisting of large deletions, insertions, duplications, inversions and translocations, were determined using Manta, Delly, SvABA, and Lumpy software. Annotations of the Manta and Delly SVs were performed using the AnnotSV program supplemented with gene enhancer data from GeneHance.
Sequence Analysis: Primers were designed encompassing the locations of mutations to amplify 100-300-bp products. PCR was performed using the Fast Cycling PCR kit (Qiagen) according to the manufacturer's recommended protocol with the following conditions: 95° C. for 5 minutes; 35 cycles of (96° C. for 5 seconds, 60° C. for 5 seconds, and 68° C. for 45 seconds) 72° C. for 1 minute. PCR products were gel purified and Sanger sequenced with the same primers used for PCR. cDNA and protein mutation nomenclature standardized by the Human Genome Variation Society (HGVS, world wide web at “hgvs.org/mutnomen”) was used to describe the mutations observed.
ImmunoblotAnalysis: Protein lysates from pleural MM cell lines were prepared using RIPA cell lysis buffer supplemented with 2 mM PMSF. Pleural MM tumor protein lysates were prepared by pulverizing frozen tumor pieces in liquid nitrogen, using a mortar and pestle, and then disrupting the cells in 1X cell lysis buffer from Cell Signaling (Danvers, MA) supplemented with 2 mM PMSF. All protein lysates were incubated for 30 minutes on ice followed by centrifugation for 20 minutes at 4° C. Bradford reagent was used to measure protein concentrations. Then, 30 μg cell lysates were loaded into Bis-Tris gels (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked with 5% non-fat milk in TBS buffer with Tween 20 for 1 hour, followed by incubation with primary antibodies at 4° C. overnight. After washing, membranes were incubated with secondary antibody at room temperature, and further washed three times. The antibodies used for immunoblotting were from Antibodies, Inc. (Davis, CA): anti-LRRK2/Dardarin (C-terminus), clone N241A/34, #75-253) and Santa Cruz Biotechnology (Dallas, TX): anti-R-actin (ACTB, sc-47778) and anti-GAPDH (sc-32233).
Mutated candidate genes were selected for further evaluation based on several criteria: 1) DNA variants present at <0.05% allele frequency in the general population, as determined using the ExacDB database, and a CADD score greater than 20; 2) the candidate gene has been previously implicated in cancer either somatically or hereditarily, or is known to be connected with a cellular process thought to be a hallmark of cancer; 3) greater priority given to mutations known to affect protein coding sequence (e.g., nonsense, indel, splice site mutations); and 4) genes mutated in more than one family. Mutations in selected candidate genes were confirmed by Sanger sequencing. Based on these criteria, germline variants were identified in 21 genes (1 involving heterozygous loss of the entire gene) that were considered to be the most promising candidates (data not shown). Eleven of the candidate genes (ATM, ATR, BRCA2, BRIP1, CHEK2, MLH3, MUTYH, POLE, POLE4, POLQ, and XRCC1) encode proteins that have roles in DNA repair, whereas four (ARID1B, DNMT3A, JARID2, and SETD1B) encode proteins involved in chromatin modification. In several MM cases, 3 or 4 candidate tumor susceptibility gene variants were identified in the same individual. Interestingly, three of the genes were found to be mutated in more than one proband: CHEK2 (cases ABS3383 and ABS2640), LRRK2 (cases ABS2640 and 946-P), and POLQ (cases ABS3460 and ABS3505). Each of these same three genes, as well as several others, were found to be mutated/deleted somatically in pleural MM tumors in The Cancer Genome Atlas (TCGA) database. The germline mutations in the present cases consisted of indel (predicted frameshift), splice site, and missense mutations as well as large exonic deletions. Among the 13 index cases reported here, 8 (61.5%) had DNA sequencing variants that are predicted to be pathogenic mutations (e.g., in/dels), which are described below.
MM Case ABS2406: This individual was found to have an indel mutation involving a member of the mismatch repair (MMR) family: MSH4 (c.719dupT; p.I1e240fs) as well as a non-frameshift deletion of 18 bp in SMARCB1 (c.56_73del; p.20_25del). The deletion in SMARCB1 is predicted to affect residues located within the DNA binding domain of the protein. In addition to MM, case ABS2406 had a rhabdomyosarcoma and a Schwannoma, and her daughter also developed more than one tumor
MI UCase ABS2640: An unusual alteration of CHEK2 was found in case ABS2640
MM Case ABS2813: In this MM patient (
MM Case ABS3425: In this individual, proband (II-2) in the pedigree shown in
MM Case ABS3460: This MM patient harbored sequencing variants in four notable cancer-related genes: POLQ, XRCC1, SETD1B, and ARID1B. Remarkably, his family history included multiple and varied carcinomas, and both he (II-5) and his brother (II-6) had MM and one or more other cancers (
Notably, annotation of ARID1B protein post-translational modifications (world wide web at “phosphosite.org”) provided evidence that Serine 802 can be phosphorylated; thus, loss of this serine in case ABS3460 could have a consequential effect on ARTDIB protein function.
MM Cases R88-T and 946-P: Lastly, in a previously reported Italian family with multiple cases of MM (
Pedigrees from the remaining 7 families without predicted pathogenic germline mutations are presented in
With the availability of TCGA data on pleural MM samples, the cBioPortal Cancer Genomics software was used to identify germline and somatic mutations affecting the candidate genes identified herein. No germline mutations were found in the TCGA dataset, whereas a small percentage of somatic mutations and other alterations affected the candidate genes were identified among the 82 MM patients for which genomic data were available (data not shown). The most frequently altered genes, BRIP1, LRRK2 and RHBDF2, occurred in 3-6% of the TCGA cases. There were 2 deep deletions and a truncating mutation in LRRK2 and 3 or 4 amplifications of RHBDF2 and BRIP1, respectively. Two deep deletions, truncating mutations, and/or gene fusions were observed in CHEK2, POLQ, and DNMT3A.
Many of the genes with germline variants that were identified have been reported in other MM patients from high-risk cancer families, although with different alterations (data not shown). Among the affected genes, germline mutations of APC, ATM, BRCA2, CHEK2, and DNMT3A have each been reported. The findings presented here demonstrate that not all MM families, high-risk cancer families with at least one MM, or apparently sporadic MM cases with cancer-related germline mutations involve the BAP1 gene, and that it is likely that there are other genes associated with MM susceptibility. Access is available to the DNA and disease history of a large, unique cohort of high-risk cancer families with at least one member having MM. WGS technology was utolozed to sequence the entire genome of 14 MM patients from 13 cancer families that do not harbor germline BAP1 mutations. In this study, mutations were identified in a number of candidate cancer-related genes that may have contributed to the high incidence of cancer, including MM, in these families. Notably, several of these candidate genes were found to be mutated in more than one family. In particular, different mutations were discovered in the POLQ, CHEK2, and LRRK2 genes in unrelated individuals.
Altogether, 10 of the 13 probands exhibited germline DNA sequencing variants in cancer-related genes, 6 of which had one or more predicted pathogenic mutations; 2 of these same 6 cases also had either a large deletion encompassing two exons of a gene or an entire gene. Three of these 6 cases had two different predicted pathogenic changes. Five of these 9 probable deleterious alterations affected genes that encode proteins involved in DNA repair (CHEK2, MUTYH, POLE4, POLQ, and XRCC1), and the other 7 genes encode proteins involved in chromatin modification (ARID1B, DNMT3A, JARID2, SETD1B, SMARCB1) or other cellular pathways: LRRK2 (2 cases) and MSH4. CHEK2 encodes cell cycle checkpoint kinase 2, a DNA repair signaling kinase downstream of ATM and ATR. The 5395-bp deletion of CHEK2 exons 9 and 10 seen in MM case ABS2640 (
Although sequencing variants were found in five genes (ARID1B, DNMT3A, JARID2, SETD1B, SMARCB1) that encode proteins that participate in chromatin modification processes, a predicted pathogenic mutation was found only for DNMT3A (case ABS3425). The protein product of the DNMT3A gene, DNA methyltransferase 3 alpha, is normally involved in CpG DNA methylation and epigenetic silencing of target genes. Interestingly, DNMT3A interacts with EZH2, a histone methyltransferase that has been previously reported to be elevated as a result of BAP1 loss in MM. Additionally, inactivating germline DNMT3A mutations have been implicated in the rare DNMT3A overgrowth syndrome, which is characterized by tall stature and other physical anomalies in affected individuals. Analysis of RNA-seq and survival findings from TCGA's MM database revealed that MM patients whose tumors had low DNMT3A mRNA expression levels had a significantly better overall survival rate. How germline mutations in DNMT3A might predispose to MM is unknown.
In case ABS2406, an MSH4 indel mutation results in a predicted frameshift mutation (p.Ile240fs). Although a member of the mismatch repair (MMR) family, MSH4 has not been found to play a role in MMR, but instead is involved in homologous recombination during meiosis. However, one study determined that MSH4 does play a role in maintaining genomic stability through its ability to suppress non-homologous end joining double strand break repair.
Interestingly, a missense mutation in MSH4 was found to co-segregate with multiple family members who collectively had three gliomas and two schwannomas. ABS2406 also possess a non-frameshift deletion within SMARCB1. The gene encodes a protein that is part of the SWI-SNF chromatin remodeling complex, and the deletion is predicted to affect residues located within the DNA binding region (p.20_25del). Germline mutations of SMARCB1 are known to be associated with schwannomatosis and rhabdoid tumor predisposition syndrome. The occurrence of a rhabdoid sarcoma and schwannoma in the present index case mirrors another study where an individual with a germline deletion of SMARCB1 developed both of these types of cancers.
Whether the development of MM in ABS2406 is related to the mutation in SMARCB1 or in combination with the MSH4 mutation remains unknown.
One highly noteworthy candidate gene discovered in the present investigation is LRRK2, which incurred a deletion at a splice site in an asbestos-exposed Italian family with multiple members afflicted by pleural MM (
In a macrodissected MM tumor sample from this family, it was possible to demonstrate homo- or hemizygosity of the LRRK2 splice site mutant allele and loss of the remaining wild type (LOH), suggesting that the mutant gene acts as a driver for MM in this unique asbestos-exposed family with an unusually high penetrance of MM.
Even among heavily exposed asbestos workers, the incidence of MM is only about 5%. As is the case for BAP1, germline mutation of LRRK2 may make individuals highly susceptible to the carcinogenic effects of asbestos. Ten different pathogenic missense mutations in this gene have been described in Parkinson disease, and germline LRRK2 G2019S missense mutations have been found in ˜10% of individuals with Parkinson's disease. Interestingly, epidemiological studies have indicated that LRRK2 G2019S carriers have an increased risk of developing cancer, including hormone-related neoplasms (prostate and breast carcinomas), colon and kidney carcinomas, as well as meningioma, the latter two also part of the BAP1-TPDS tumor spectrum.
LRRK2's role in predisposing to various cancers may be due to its involvement in the DNA damage response. Treatment of mouse embryonic fibroblasts (MEFs) with the DNA damaging agent adriamycin resulted in phosphorylation of the LRRK2 protein at several sites. In contrast, LRRK2 phosphorylation was not increased in Atm knockout MEFs, indicating that LRRK2 is downstream of Atm. In addition, induction of p53 and p21 expression caused by adriamycin treatment was suppressed when LRRK2 was silenced by siRNA. Another group demonstrated that LRRK2 can phosphorylate p53, leading to its translocation to the nucleus and induction of p21 gene expression. The LRRK2 mutation identified in this family is unique in that it is a 10-bp deletion encompassing the end of exon 36, as well as the adjacent splice site and intron. It is unknown what cDNA and protein product the mutation would produce, if any. After observing loss of the wild type allele of LRRK2 in a MM tumor (
Various modifications of the described subject matter, in addition to those described herein, will be 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 (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, and the like) cited in the present application is incorporated herein by reference in its entirety and for all purposes.
This invention was made with government support under Grant Nos. CA175691 and CA06927 awarded by the National Cancer Institute. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/25469 | 4/20/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63177189 | Apr 2021 | US |
