WO2014138586A1

WO2014138586A1 - Compositions and methods for the diagnosis and treatment of dolichol deficiency related disorders

Info

Publication number: WO2014138586A1
Application number: PCT/US2014/021763
Authority: WO
Inventors: Ziqiang Guan; Rong Wen; Byron L. LAM
Original assignee: Duke University; University Of Miami
Priority date: 2013-03-08
Filing date: 2014-03-07
Publication date: 2014-09-12

Abstract

The present disclosure provides methods of treating, ameliorating and/or preventing diseases characterized by dolichol deficiency in humans by administering a therapeutically effective amount of a squalene synthase inhibitor. The present disclosure further provides biomarkers, for identifying those individuals at risk for, or suffering from, a disease characterized by a dolichol deficiency.

Description

COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF DOLICHOL DEFICIENCY

RELATED DISORDERS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent application number

61/774,708 filed March 8, 2013, the disclosure of which is hereby incorporated by reference in its entirety.

FEDERAL FUNDING LEGEND

[0002] The invention was made with government support under Grant No.'s:

5U546M069338-09 and 01 EY018586 awarded by the National Institute of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

[0003] The presently disclosed su bject matter relates to compositions and methods for the diagnosis and treatment of dolichol deficiency related disorders.

BACKGROUND

[0004] Dolichol deficiency has been recently recognized as a cause of a su btype of retinitis pigmentosa (RP). RP is a group of genetically heterogeneous retinal degenerative disorders with the prevalence of 1 in 3,000-4,500 people (15-17,20). Patients with RP experience progressive

photoreceptor death, which leads to visual impairment and total blindness in some cases.

[0005] One cause of RP has been found to be d ue to a mutation of the dehydrodolichol diphosphate synthase (DH DDS) gene. DHDDS encodes dehydrodolichol diphosphate synthase (DHDDS), a cis-prenyltransferase and a key enzyme in dehydrodolichol diphosphate biosynthesis (5-7). Using whole-exome sequencing, a single-nucleotide mutation, c. 124A>G was identified in the DHDDS gene that causes the retinal degeneration in a family with autosomal recessive RP (13). The mutation results in a change of a highly conserved residue Lys42 to Glu. The same mutation was subsequently confirmed as the cause of RP in 12% of arRP patients of AJ origin and is found heterozygously in 1 out of 322 in the AJ population (14). Mutations in more than 60 genes are implicated in RP, including in nearly 50% of arRP cases (15-17).

[0006] Dolichols are polyisoprenold alcohols first identified in 1960 (3). Among the largest lipids in the cell, these linear polymers of isoprene normally contain 14 to 24 isoprene units with a saturated a-isoprene unit (see FIG. 1). While the phosphorylated derivative of dolichol, dolichyl phosphate, has an established role as a lipid carrier of oligosaccharide in the biosynthesis of N-linked protein glycosylation, the function(s) of the free alcohol and its esterified form is much less clear (16). Studies on model membranes and biological membranes showed that dolichols can greatly influence the fluidity and increase membrane permeability (21-23). It is also believed that dolichols serve as inducers of vesicle fusion (24).

[0007] The initial steps of biosynthesis of dolichol, cholesterol, and ubiquinone follow a common pathway known as the mevalonate pathway (25). As shown in FIG. 2, at the branching point of farnesyl-pyrophosphate (FPP), cis-cis addition of isopentenyl-pyrophosphate (IPP) produces dolichols. Therefore, disturbance of the mevalonate pathway would influence the flux of dolichol biosynthesis. For example, feeding mice with high-cholesterol diet to suppress 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) significantly decreases the dolichol levels in the liver (30). On the other hand, inhibition of squalene synthase increased the FPP as well as dolichol levels in rat liver (26). Inhibition of the HMG-CoA reductase by statins used widely for the treatment of hypercholesterolemia has been documented (27). Squalene synthase inhibitors, which work further downstream of the cholesterol mevalonate pathway after the branching point, have also been considered for treating hypercholesterolemia (28). In fact, phase III clinical trials had been conducted to use lapaquistat acetate, a squalene tynthase inhibitor, for hypercholerterolemia (29).

[0008] Accordingly, there remains an unmet need for improved methods for diagnosing and treating retinitis pigmentosa. The present disclosure provides such improved methods. SUMMARY

[0009] One aspect of the present disclosure provides methods of treating disease characterized by dolichol deficiency in a subject comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a squalene synthase inhibitor such that the FPP pool is increased and the disease is treated.

[00010] Another aspect of the present disclosure provides methods of preventing and/or ameliorating a disease characterized by dolichol deficiency in a subject comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a squalene synthase inhibitor such that the FPP pool is increased and the disease is prevented.

[00011] In some embodiments, the squalene synthase inhibitor is selected from the group consisting of zaragozic acid, lapaquistat acetate, E -27865, 2,8-dioxabicyclo[3,2,l]-octane and derivatives thereof, dicarboxylic acid, quinuclidine derivatives, 4,1-benzoxazepine, substituted morpholine derivatives, phenoxypropylamines, monooxygenase, synthase, combinations there and pharmaceutical compositions thereof.

[00012] In another embodiment, the disease is characterized by a mutation in the DHDDS gene.

In certain embodiments, the disease comprises retinitis pigmentosa.

[00013] In yet another embodiment, the squalene synthase inhibitor is administered at the onset of disease. In other embodiments, the squalene synthase inhibitor is administered prior to disease symptoms. In yet other embodiments, the squalene synthase inhibitor is administered after the onset of disease symptoms.

[00014] Another aspect of the present disclosure provides a method for identifying a subject with a DHDDS mutation comprising, consisting of, or consisting essentially of (1) obtaining a biological sample from the subject; (2) determining at least one dolichol ratio in the sample; (3) comparing at least one dolichol ratio against that of at least one control; and (4) administering a squalene synthase inhibitor in accordance with the present disclosure if at least one of the ratios in the biological sample are higher than that of the control.

[00015] In some embodiments, the dolichol ratios measured comprise dolichol 17:dolichol 19; dolichol 18:dolichol 19; dolichol 17:dolichol 20; dolichol 18:dolichol 20; and com binations thereof.

[00016] In some embodiments, the biological sample is selected from the group consisting of whole blood, plasma, serum, urine, saliva and tissue biopsy (e.g., a somatic cell). In certain em bodiments, the biological sample comprises a blood sample. In other embodiments, the biological sample is urine.

[00017] Yet a nother aspect of the present disclosure provides a kit comprising, consisting of, or consisting essentially of a reagent capa ble of detecting the dolichol ratios as described herein and a sheet of instructions capa ble of diagnosing the presence of a DHDDS mutation.

[00018] Yet a nother aspect of the present disclosure provides for all that is disclosed and illustrated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[00019] The foregoing aspects and other features of the invention are explained in the following description, taken in connection with the accompanying drawings, wherein:

[00020] FIG. 1 is a schematic showing the structure of dolichol. In eukaryotic cells, dolichols are made up of varying num bers (n, 14 to 24) of isoprene units with an a-saturated isoprene unit and two trans units at the ω-end of the chain.

[00021] FIG. 2 is a schematic showing the mevalonate pathway and strategy of increasing dolichol levels by inhibiting squalene synthesis according to one or more embodiments of the presently disclosed su bject matter. Dolichol, cholesterol, and u biquinone share the same biosynthetic pathway to the branching point of farnesyl PP (FPP). Inhibition of squalene synthase slows further steps of cholesterol and the accumulation of FPP can lead to an increase in the production of dolichol according to one or more em bodiments of the presently d isclosed su bject matter.

[00022] FIG. 3 is a graph showing dolichol-17 (Dol-17), dolichol-18 (Dol-18), dolichol-19 (Dol-19), and dolichol-20 (Dol-20) levels in fibroblasts from 3 patients with K42E DH DDS treated with squalane synthase inhibitor zaragozic acid (Treated) compared to untreated cells (Control) and showing statistically significant increases in dolichol levels in treated cells according to one or more embodiments of the presently disclosed su bject matter. Asterisks, P<0.05; dou ble asterisks, P<0.01. [00023] FIG.'s 4A - 4F are graphs showing the blood dolichol ratios in unaffected normal individuals (UA), carriers (CI, C2), and patients with DHDDS mutation (Ptl, Pt2, and Pt3) according to one or more embodiments of the presently disclosed subject matter. A) Dolichol 17:dolichol 19 ratio; B) Dolichol 18:dolichol 19 ratio; C) Comparison of dolichol ratios 18:19 and 17:19; D) Dolichol 17:dolichol 20 ratio; E) Dolichol 18:dolichol 20 ratio; and F) Dolichol 19:dolichol 20 ratio.

[00024] FIG.'s 5A-5C show dolichol analysis of the primary DHDDS RP family used for FIG 4 according to one or more embodiments of the presently disclosed subject matter. A) A schematic showing the genetic tree of the primary DHDDS RP family; B) A graph showing the blood plasma ratio of DOL-18:Dol-19 in each individual of the family; and C) A chromatography reading showing the amounts of Dol-17, Dol-18, Dol-19 and Dol-20 in blood plasma for in each individual of the family.

[00025] FIG.'s 6A-6E are graphs showing Dol-18/Doll9 ratio analysis in plasma and urine from normal, K42E carriers, and K42E/K42E patients according to one or more embodiments of the presently disclosed subject matter. A) Plasma dolichol profiling was performed on 6 K42E/K42E patients, 25 K42E carriers, 16 normal individuals. B) ROC curve analysis showing that plasma D18/D19 ratios discriminate patients from carriers with 100% sensitivity and specificity and C) patients from normal individuals with 100% sensitivity and specificity; D) Urinary dolichol profiling was performed on 6 K42E/K42E patients, 30 K42E carriers, and 13 normal individuals. E) Urinary D18/D19 ratios also discriminate patients from carriers with 100% sensitivity and specificity and F) carriers from normal individuals with 100% sensitivity and specificity. A bar representing the mean D18/D19 ratio (± SD) of each group is next to the plotted data (A and D). D18/D19 ratios are well separated among the three genotypic groups, with the separation being greater in urine than in plasma. The dotted diagonal lines indicate a reference line of AUC = 0.5 (B, C, E, and F).

[00026] FIG. 7 is a graph showing the Dol-18/Doll9 ratio in urine from K42E/K42E (Affected),

K42E (Carrier), and wt (Normal) patients indicating that urine samples can be used to assess dolichol ratios from patients according to one or more embodiments of the presently disclosed subject matter. The urinary D18/D19 ratios of the affected patients are much higher than in the normal individuals, and the ratios of the K42E carriers are lower than those of the patients but higher than the normal. [00027] FIG. 8 is a bar graph showing the ratio of Dol-18: Dol-19 obtained from urine samples of

12 unaffected (WT), 15 K42E (Carrier) and 6 K42E/K42E (Affected) individuals according to one or more embod iments of the presently disclosed su bject matter. The u rinary D18/D19 ratios of the affected patients are much higher than in the unaffected individuals, whereas the D18/D19 ratios of the K42E carriers are lower than those of the patients but higher than the u naffected.

DETAILED DESCRIPTION

[00028] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred em bodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the d isclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[00029] Articles "a" and "an" are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and can include more than one element.

[00030] Unless otherwise defined, all technical terms used herein have the same meaning as commonly u nderstood by one of ordinary skill in the art to which this d isclosure belongs.

[00031] The present inventors have previously identified a single-nucleotide mutation in the dehydrodolichol diphosphate syntase-encoding DHDDS gene as the cause of autosomal recessive retinitis pigmentosa (ar P) in a family of Ashkenazi Jewish (AJ) origin (13). The same mutation was su bsequently confirmed as the cause of RP in 12% of arRP patients of AJ origin and is fou nd heterozygously in 1 out of 322 in the AJ population (14). The inventors postulated that inhibiting squalene synthase could increase the farnesyl diphosphate (FPP) pool and thus increase the production of dolichol by the mutant DH DDS. In one aspect, the present disclosure provides compositions and methods for treating diseases related to dolichol deficiency by inhibiting squalene synthase. In one aspect, the present disclosure provides methods for identifying a su bject with a DH DDS mutation and other d iseases in which dolichol biosynthesis is altered. [00032] One aspect of the present disclosure provides a method of treating, ameliorating and/or preventing a disease characterized by dolichol deficiency in a subject comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a squalene synthase inhibitor such that the disease is treated and or prevented.

[00033] As used herein, the term "subject" is intended to include human and non-human animals. Exemplary human subjects include a human patient suffering from a dolichol deficiency, such as one characterized by a mutation in the DHDDS gene, e.g., retinitis pigmentosa ( P). The term

"non-human animals" includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals (such as sheep, dogs, cats, cows, pigs, etc.), and rodents (such as mice, rats, hamsters, guinea pigs, etc.).

[00034] As used herein, the term "disease characterized by a dolichol deficiency" refers to any disease caused by a disruption of the mevalonate pathway such that the biosynthesis of dolichol is disrupted or blocked. In some embodiments, the disease comprises at least one mutation in the DHDDS gene. Such diseases include, but are not limited to, retinitis pigmentosa (RP).

[00035] As used herein, the term "squalene synthase inhibitor" refers to those compounds that are capable of modulating (e.g., blocking, disrupting, etc.) squalene synthase (SQS), also known as Farnesyl-diphosphate farnesyltransfersase (FDFTl), thereby preventing the condensation of two farnesyl diphosphate (FPP) molecules to form presqualene diphosphate (PSPP), and the reduction of PSPP to produce squalene. Examples of such compounds include, but are not limited to, zaragozic acid (see, e.g., Kourounakis, AP et al 2011 Curr Med Chem 18:4418-4439, the contents of which are hereby

incorporated by reference in its entirety), lapaquistat acetate, ER-27865, 2,8-dioxabicyclo[3,2,l]-octane and derivatives thereof (such as those described in Kourounakis, AP et al 2011 supra), dicarboxylic acid, quinuclidine derivatives (such as those described in Kourounakis, AP et al 2011 supra),

4,1-benzoxazepine, substituted morpholine derivatives (such as those described in Kourounakis, AP et al 2011 supra), phenoxypropylamines (such as those described in Brown, G. et al. 1995 J. Med. Chem. 38:4157-4160, the contents of which is hereby incorporated by reference in its entirety),

monooxygenase, synthase, combinations thereof and pharmaceutical compositions thereof. [00036] As used herein, the terms "treat" and "prevent" are not intended to be absolute terms.

Treatment can refer to any reduction in the frequency or severity of symptoms, amelioration of symptoms, improvement in patient comfort and/or function, etc. The effect of treatment can be compared to an individual or pool of individuals not receiving a given treatment, or to the same patient prior to, or after cessation of treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques. The term "prevent" refers to a decrease in the occurrence of symptoms of a disease or disorder characterized by dolichol deficiency in a patient. As indicated above, the prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur a bsent treatment.

[00037] As used herein, the term "therapeutically effective" refers to a dosage of a compound

(e.g., a squalene synthase inhibitor) that is effective for eliciting a desired effect. This term as used herein may also refer to an amount effective at bringing about a desired in vivo effect in an animal, mammal, or human, such as increasing the FPP pool and the like. A therapeutically effective amount may be administered in one or more administrations (e.g., the compound may be given as a

preventative treatment or therapeutically at any stage of disease progression, before or after symptoms, and the like), applications or dosages and is not intended to be limited to a particular formulation, combination or administration route. It is within the scope of the present disclosure that the squalene synthase inhibitor may be administered at various times during the course of disease in the subject. The times of administration and dosages used will depend on several factors, such as the goal of treatment (e.g., treating v. preventing), condition of the subject, etc. and can be readily determined by one skilled in the art. For example, in one embodiment the squalene synthase inhibitor is administered at the onset of disease. In other embodiments, the squalene synthase inhibitor is administered after onset of the disease. In yet other embodiments, the mast cell modulator is administered prior to the onset of disease. [00038] The term "administration" or "administering," as used herein, refers to providing, contacting, and/or delivery of a squalene synthase inhibitor compound by any appropriate route to achieve the desired effect. These compounds may be administered to a subject in numerous ways including, but not limited to, orally, ocularly, nasally, intravenously, topically, as aerosols, suppository, etc. and may be used in combination.

[00039] In certain embodiments, the squalene synthase inhibitor may be in the form of a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to the combination of compound (i.e., for example, squalene synthase inhibitor) with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, in vivo or ex vivo. A "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants. (See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975)).

[00040] As used herein, the term "pharmaceutically acceptable salt" refers to any

pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present disclosure which, upon administration to a su bject, is capable of providing a compound of this invention or an active metabolite or residue thereof. "Salts" of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. [00041] Examples of bases include, but are not limited to, alkali metals (e.g., sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides, ammonia, and compounds of formula NW₄ ⁺, wherein W is Ci_₄ alkyl, and the like.

[00042] Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate,

cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄ ⁺ (wherein W is a Ci_₄ alkyl group), and the like. For therapeutic use, salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically accepta ble may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[00043] The present inventors have discovered that a mutation in the gene encoding the DHDDS enzyme that changes Lys42 to Glu42 leads not only to lower dolichol levels, but also to a characteristic blood dolichol profile. In one aspect of the present disclosure, the inventors measured dolichols (Dol-17, Dol-18, Dol-19, and Dol-20) in blood plasma by liquid chromatography coupled with mass spectrometry (LC/MS) and demonstrated the characteristic dolichol profile. The characteristic dolichol profile is described in Example 2 and FIG.'s 3-8.

[00044] Hence, another aspect of the present disclosure provides a method for identifying a subject with a DHDDS mutation comprising, consisting of, or consisting essentially of (1) obtaining a biological sample from the subject; (2) determining at least one dolichol ratio in the sample; (3) comparing the at least one dolichol ratio against that of at least one control; and (4) administering a squalene synthase inhibitor in accordance with the present disclosure if at least one dolichol ratio in the biological sample is significantly lower than that of the control. [00045] As used herein, the term "significantly" refers to anything that is above a control (e.g., the amount found in an individual who does not have the DHDDS mutation). For example, dolichol ratios may be 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6. fold, 0.7 fold, 0.8 fold, 0.9 fold, 1 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6. fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6. fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, or higher than that of a control.

[00046] As used herein, the term "dolichol" refers to any of a group of long-chain mostly unsaturated organic compounds that are made up of varying numbers of isoprene units terminating in an a-saturated isoprenoid group, containing an alcohol functional group and synthesized in the mevalonic pathway. Suitable dolichols include, but are not limited to, dolichol 17, dolichol 18, dolichol 19 and dolichol 20. As described herein, the DHDDS mutation leads not only to lower dolichol levels, but a characteristic blood dolichol profile. In some embodiments, the dolichol ratios measured comprise dolichol 17:dolichol 19; dolichol 18:dolichol 19; dolichol 17:dolichol 20; dolichol 18:dolichol 20; and combinations thereof.

[00047] In some embodiments, the dolichol levels are detected in a biological sample. As used herein, the term "biological sample" refers to any sample that can be taken from a subject in which the presence or a bsence of the biological marker can be determined. Suitable examples of biological samples include, but are not limited to, whole blood, plasma, serum, urine, saliva and tissue biopsy (e.g., a somatic cell). In certain embodiments, the sample comprises blood. In other embodiments, the sample comprises urine.

[00048] Other embodiments of the present disclosure provides kits for the detection, characterization, and diagnosis of a disease characterized by dolichol deficiency in a su bject. In some embodiments, the kit comprises, consists of, or consists essentially of a reagent capable of detecting dolichol levels and a sheet of instructions capable of diagnosing the presence of a DHDDS mutation.

[00049] In one embodiment of the presently disclosed subject matter a method is provided for identifying a subject with a dehydrodolichol diphosphate synthase (DHDDS) mutation comprising:

obtaining a biological sample from the subject; determining at least one dolichol ratio in the sample; and comparing the at least one dolichol ratio against that of a control, wherein the subject is identified as having the DHDDS mutation if at least one of the ratios in the biological sample is higher than that of the control.

[00050] The method for identifying a subject with a DHDDS mutation can further comprise administering a squalene synthase inhibitor if at least one of the ratios in the biological sample is higher than that of the control.

[00051] In the method for identifying a subject with a DHDDS mutation, the dolichol ratio can be selected from the group consisting of dolichol 17:dolichol 19; dolichol 18:dolichol 19; dolichol

17:dolichol 20; dolichol 18:dolichol 20; dolichol 19:dolichol 20 and combinations thereof. The dolichol ratio can comprise dolichol 18:dolichol 19. The dolichol ratio can consist of dolichol 18:dolichol 19.

[00052] In the method for identifying a subject with a DHDDS mutation, the biological sample can be selected from the group consisting of whole blood, plasma, serum, urine, saliva, and tissue biopsy. The biological sample can comprise urine. The biological sample can comprise whole blood.

[00053] In one embodiment of the presently disclosed subject matter, a method is provided for identifying a subject having a dehydrodolichol diphosphate synthase (DHDDS) mutation, the method comprising: determining at least one dolichol ratio in a biological sample derived from a subject; and identifying the subject as having a DHDDS mutation if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

[00054] In the method for identifying a subject having a DHDDS mutation, the dolichol ratio can comprise one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20. The dolichol ratio can comprise dolichol

18:dolichol 19. The dolichol ratio can consist of dolichol 18:dolichol 19.

[00055] In the method for identifying a subject having a DHDDS mutation, the biological sample can comprise whole blood, plasma, serum, urine, saliva, or tissue biopsy. The biological sample can comprise urine. The biological sample can comprise plasma. The dolichol ratio can comprise dolichol 18:dolichol 19 and the biological sample can comprise plasma or urine. The dolichol ratio can comprise dolichol 18:dolichol 19, the biological sample can comprise plasma or urine, and the dolichol 18:dolichol 19 ratio can be greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

[00056] In the method for identifying a subject having a DHDDS mutation, the method can further comprise administering a squalene synthase inhibitor if at least one of the ratios in the biological sample are higher than that of the control.

[00057] In the method for identifying a subject having a DHDDS mutation, the determining the at least one dolichol ratio can be carried out by liquid chromatography-mass spectrometry (LC-MS).

[00058] In one embodiment of the presently disclosed subject matter, a method is provided for identifying a subject having a dolichol deficiency, the method comprising: determining at least one dolichol ratio in a biological sample derived from a subject; and identifying the subject as having a dolichol deficiency if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

[00059] In the method for identifying a subject having a dolichol deficiency, the dolichol ratio can comprise one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol

17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20. The dolichol ratio can comprise dolichol 18:dolichol 19. The dolichol ratio can consist of dolichol 18:dolichol 19.

[00060] In the method for identifying a subject having a dolichol deficiency, the biological sample can comprise whole blood, plasma, serum, urine, saliva, or tissue biopsy. The biological sample can comprise urine. The biological sample can comprise plasma. The dolichol ratio can comprise dolichol 18:dolichol 19 and the biological sample can comprise plasma or urine. The dolichol ratio can comprise dolichol 18:dolichol 19, the biological sample can comprise plasma or urine, and the dolichol 18:dolichol 19 ratio can be greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

[00061] In the method for identifying a subject having a dolichol deficiency, the method can further comprise administering a squalene synthase inhibitor if at least one of the ratios in the biological sample are higher than that of the control.

[00062] In the method for identifying a subject having a dolichol deficiency, the determining the at least one dolichol ratio can be carried out by liquid chromatography-mass spectrometry (LC-MS). [00063] In one embodiment of the presently disclosed subject matter, a method is provided for identifying a subject that has altered dolichol biosynthesis, the method comprising: determining at least one dolichol ratio in a biological sample derived from a subject; and identifying the subject as having altered dolichol biosynthesis if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

[00064] In the method for identifying a subject that has altered dolichol biosynthesis, the dolichol ratio can comprise one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20. The dolichol ratio can comprise dolichol 18:dolichol 19. The dolichol ratio can consist of dolichol 18:dolichol 19.

[00065] In the method for identifying a subject that has altered dolichol biosynthesis, the biological sample can comprise whole blood, plasma, serum, urine, saliva, or tissue biopsy. The biological sample can comprise urine. The biological sample can comprise plasma. The dolichol ratio can comprise dolichol 18:dolichol 19 and the biological sample can comprise plasma or urine. The dolichol ratio can comprise dolichol 18:dolichol 19, the biological sample can comprise plasma or urine, and the dolichol 18:dolichol 19 ratio can be greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

[00066] In the method for identifying a subject that has altered dolichol biosynthesis, the method can further comprise administering a squalene synthase inhibitor if at least one of the ratios in the biological sample are higher than that of the control.

[00067] In the method for identifying a subject that has altered dolichol biosynthesis, the determining the at least one dolichol ratio can be carried out by liquid chromatography-mass spectrometry (LC-MS).

[00068] In one embodiment of the presently disclosed subject matter a kit is provided for identifying a subject having a dehydrodolichol diphosphate synthase (DHDDS) mutation, the kit comprising: one or more reagents for lipid extraction for determining at least one dolichol ratio in a biological sample derived from a subject; and instructions for carrying out the determination of the at least one dolichol ratio in the biological sample and for identifying the subject as having a DHDDS mutation if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

[00069] In the kit for identifying a subject having a DHDDS mutation, the dolichol ratio can comprise one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20. The dolichol ratio can comprise dolichol

18:dolichol 19. The dolichol ratio can consist of dolichol 18:dolichol 19.

[00070] In the kit for identifying a subject having a DHDDS mutation, the biological sample can comprise whole blood, plasma, serum, urine, saliva, or tissue biopsy. The biological sample can comprise urine. The biological sample can comprise plasma. The dolichol ratio can comprise dolichol 18:dolichol 19 and the biological sample can comprise plasma or urine. The dolichol ratio can comprise dolichol 18:dolichol 19, the biological sample can comprise plasma or urine, and the dolichol 18:dolichol 19 ratio can greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

[00071] In the kit for identifying a subject having a DHDDS mutation, the determining the at least one dolichol ratio can be carried out by liquid chromatography-mass spectrometry (LC-MS).

[00072] In the kit for identifying a subject having a DHDDS mutation, the kit can further comprise a sample collection kit for the biological sample.

[00073] In one embodiment of the presently disclosed subject matter, a kit is provided for identifying a subject having a dolichol deficiency, the kit comprising: one or more reagents for lipid extraction for determining at least one dolichol ratio in a biological sample derived from a subject; and instructions for carrying out the determination of the at least one dolichol ratio in the biological sample and for identifying the subject as having a having a dolichol deficiency if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

[00074] In the kit for identifying a subject having a dolichol deficiency, the dolichol ratio can comprise one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20. The dolichol ratio can comprise dolichol

18:dolichol 19. The dolichol ratio can consist of dolichol 18:dolichol 19. [00075] In the kit for identifying a subject having a dolichol deficiency, the biological sample can comprise whole blood, plasma, serum, urine, saliva, or tissue biopsy. The biological sample can comprise urine. The biological sample can comprise plasma. The dolichol ratio can comprise dolichol 18:dolichol 19 and the biological sample can comprise plasma or urine. The dolichol ratio can comprise dolichol 18:dolichol 19, the biological sample can comprise plasma or urine, and the dolichol 18:dolichol 19 ratio can be greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

[00076] In the kit for identifying a subject having a dolichol deficiency, the determining the at least one dolichol ratio can be carried out by liquid chromatography-mass spectrometry (LC-MS).

[00077] In the kit for identifying a subject having a dolichol deficiency, the kit can further comprise a sample collection kit for the biological sample.

[00078] In one embodiment of the presently disclosed subject matter, a kit is provided for identifying a subject that has altered dolichol biosynthesis, the kit comprising: one or more reagents for lipid extraction for determining at least one dolichol ratio in a biological sample derived from a subject; and instructions for carrying out the determination of the at least one dolichol ratio in the biological sample and for identifying the subject as having altered dolichol biosynthesis if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

[00079] In the kit for identifying a subject that has altered dolichol biosynthesis, the dolichol ratio can comprise one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20. The dolichol ratio can comprise dolichol 18:dolichol 19. The dolichol ratio can consist of dolichol 18:dolichol 19.

[00080] In the kit for identifying a subject that has altered dolichol biosynthesis, the biological sample can comprise whole blood, plasma, serum, urine, saliva, or tissue biopsy. The biological sample can comprise urine. The biological sample can comprise plasma. The dolichol ratio can comprise dolichol 18:dolichol 19 and the biological sample can comprise plasma or urine. The dolichol ratio can comprise dolichol 18:dolichol 19, the biological sample can comprise plasma or urine, and the dolichol 18:dolichol 19 ratio can be greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

[00081] In the kit for identifying a subject that has altered dolichol biosynthesis, the determining the at least one dolichol ratio can be carried out by liquid chromatography-mass spectrometry (LC-MS).

[00082] In the kit for identifying a subject that has altered dolichol biosynthesis, the kit can further comprise a sample collection kit for the biological sample.

[00083] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1

Increase in the FPP pool by inhibition ofsqualene synthase increases the production of dolichol by mutant

DHDDS.

[00084] It was hypothesized that to increase the FPP pool by inhibiting squalene synthase could increase the production of dolichol by the mutant DHDDS. FIG. 2 is a schematic showing the mevalonate pathway and strategy of increasing dolichol levels by inhibiting squalene synthesis according to embodiments of the present disclosure. Dolichol, cholesterol, and u biquinone share the same biosynthetic pathway to the branching point of farnesyl PP (FPP) as can be seen in FIG. 2. The present inventors postulated that inhibition of squalene synthase could slow further steps of cholesterol and the accumulation of FPP could lead to an increase in the production of dolichol.

[00085] An experiment was performed as follows. Fibroblasts from 3 patients with the K42E

DHDDS mutation were treated with squalene synthase inhibitor zaragozic acid (0.1 mM) for 4 days. Cells were harvested and total lipids were extracted. Dolichol levels were measured by liquid chromatography coupled with mass spectrometry (LC/MS). The dominant dolichol species observed were Dol-17 and Dol-18. As shown in FIG. 3, inhibition of squalene synthase significantly increased the levels of Dol-17, Dol-18 and Dol-20. Paired Student t test was used to compare the levels of each dolichol species between control and treated cells. Asterisks, P<0.05; double asterisks, P<0.01. See Table 1 below for details. Ta&te 1, Dotiehol (eveis in fitebtasts from p ienis wiih K42E DHDDS mttt ari

These results suggest that squalene synthase inhibitors, including but not limited to, zaragozic acid, lapaquistat acetate and the like can be used to treat diseases caused by dolichol deficiency, including but not limited to, retinal degeneration associated with DHDDS mutations.

Example 2

Blood dolichol ratios in unaffected normal individuals, carriers and patients with DHDDS mutations.

[00086] As described herein above, the DHDDS gene encodes a key enzyme, DHDDS

(dehydrodolichol diphosphate biosynthesis) and a single amino acid mutation in this enzyme (K42E) leads to lower dolichol levels. This DHDDS mutation was shown to cause 12-20% of autosomal recessive P patients of Ashkenazi Jewish (AJ) origin. The present inventors measured dolichols (Dol-17, Dol-18, Dol-19, and Dol-20) in wild type (wt), carrier (K42E), and affected (K42E/ K42E) individuals by liquid chromatography coupled with mass spectrometry (LC/MS) as described herein below.

[00087] Materials and Methods.

[00088] Sample collection. This study was approved by the Institutional Review Board of the

University of Miami (protocol number 20110767). All participants provided written informed consent. Sample collection was untimed and without restrictions, including diet and liquid intake. Blood was collected in Vacutainer plasma preparation tubes (PPT) (5 ml capacity, BD, Franklin Lakes, NJ), spun at 1,300 g for 10 min. Plasma was obtained and stored at -80°C. Off-site plasma samples were collected by designated labs in the vicinity of residences of the individuals and shipped at ambient temperature via express carriers to the Bascom Palmer Eye Institute in Miami, FL.

[00089] Urine samples (20-30 ml) were collected in 50 ml tu bes containing sodium azide (~0.02% final concentration) and stored at -80°C. Sample collection kits were provided to individuals for off-site collection at their residences. Samples were shipped at ambient temperature via express carriers to the Bascom Palmer Eye Institute.

[00090] Lipid extraction. Lipid extraction was done following a modified Bligh and Dyer method (18 ). Briefly, plasma (0.3 ml) was mixed with methanol (0.3 ml) on a BULLET BLENDER (Next Advance, Averill Park, NY) for 2 min. Chloroform (0.3 ml) was then added and mixed again for 2 min. Samples were subsequently centrifuged for 10 min, and the lower chloroform layer containing lipids was transferred to a new tube. Lipids (in chloroform) were dried for 2 h or overnight on a SpeedVac (Savant Instruments, Holbrook, NY), flushed with argon, and stored at -20°C. For urinary samples, 3 ml of urine was mixed with 3 ml of methanol by vortexing for 1 min. Chloroform (1.5 ml) was added and vortexed again for 1 min. The sample was then centrifuged for 10 min, and the lower chloroform layer was transferred to a new tube. Lipids were dried on a Speed- Vac, flushed with argon, and stored at -20°C, as described above. Dried lipids were shipped via express carriers at ambient temperature to Duke University in Durham, NC for dolichol analysis.

[00091] LC-MS analysis of dolichols. Dolichols were analyzed by LC-MS (19) performed in multiple reaction monitoring (M RM) mode using a SHIMADZU LC system (comprising a solvent degasser, two LC-IOA pumps, and a SCL- 10A system controller) coupled to a 4000 Q-TRAP HYBRID TRIPLE quadrupole linear ion-trap mass spectrometer equipped with a Turbo V ion source (AB-Sciex, Foster City, CA). LC was operated at a flow rate of 200 μΙ/min with a linear gradient as follows: 100% of mobile phase A was held isocratically for 2 min, and then linearly increased to 100% mobile phase B over 14 min, and held at 100% B for 4 min. Mobile phase A consisted of methanol/acetonitrile/aqueous 1 mM ammonium acetate (60/20/20, v/v/v). Mobile phase B consisted of 100% ethanol containing 1 mM ammonium acetate. A ZORBAX SB-C8 reversed-phase column (5 μιη, 2.1 ^χ 50 mm) was obtained from AGILENT.

[00092] MRM was performed in the negative ion mode with MS settings as follows: curtain gas

(CUR) = 20 psi (pressure); gas-1 (GS1) = 20 psi; gas-2 (GS2) = 30 psi; ion spray (IS) voltage = -4500 V; source temperature (TEM) = 350°C; interface heater = ON; declustering potential (DP) = -40V; entrance potential (EP) = -10V; and CXP = -5V. The voltage used for collision-induced dissociation was -40V (laboratory frame of energy). Nitrogen was used as the collision gas. The MRM pairs for D17, D18, D19, and D20 were 1236.2/59, 1304.2/59, 1372.2/59, and 1440.3/59, respectively. In these M M pairs, the precursor ions are the [M+acetate]^" adduct ions, and the product ions are the acetate ions (m/z 59).

[00093] Statistical analysis. All statistical analyses, including i -test, ANOVA, and receiver operating characteristic (ROC) curve analysis, were performed using IBM SPSS Statistics software (IBM, Armonk, NY). Significant differences in the mean values between groups were analyzed by Student t -test for two groups or by ANOVA (ANOVA) and Tukey test for more than two groups. Data are presented as mean ± SD. Cutoff levels were selected from the table of ROC curve coordinates to obtain the highest sensitivity and specificity.

[00094] The demographics, DHDDS genotypes, and dolichol profiling data of all study subjects are summarized in Table 2.

Table 2. Demographics, DHDDS genotypes, and D18/D19 ratios in plasma and urine

a Race and ethnicity were self-reported.

b Data are presented as mean ± SD.

[00095] Plasma and urinary dolichol profiles in a family with the K42E DHDDS mutation. As shown in FIG. 4, calculation of the ratios of Dol-17/Dol-19, Dol-18/Dol-19, Dol-17/Dol-20, Dol-18/Dol-20 and Dol-19/Dol-20 reveals striking difference between the unaffected sibling (UA), carriers (CI, C2) and the affected patients (Ptl, Pt2, and Pt3). In UA, the Dol-18:Dol-19 ratio is near 0.5, but it is more than 1.5 in the affected patients. The Dol-18:Dol-19 ratio is close to 1 in UA, but is well a bove 2 in the affected patients. The ratios in the carriers are in between. The alteration of dolichol profiles by the mutation is best described by these ratios.

[00096] FIG. 5A shows the genetic tree of the sentinel DH DDS P family used for FIG 4. FIG.'s

5B and 5C are a graph and a chromatography reading, respectively, showing the ratio of DOL-18:Dol-19 and the amounts of Dol-17, Dol-18, Dol-19 and Dol-20 in urine from each individual of the family, respectively. In the three affected siblings, dolichol profiles are consistently shorter (by a bout one isoprene u nit) than that of the unaffected sibling who has the wild type (WT) DHDDS. In the affected ind ividuals, D18 became the domina nt dolichol species (FIG. 5C). The profiles of two carriers (parents) are longer than those of the affected ind ividuals but shorter than that of the normal individual, suggesting that both the WT and mutant DH DDS proteins are functional (FIG. 5C). The separations in the D18/D19 ratios among the affected individuals, carriers, and unaffected individual are greater in urine than in plasma (data not shown).

[00097] Plasma and urinary D18/D19 ratios in a rRP patients with the K42E mutation, in carriers, and in normal individuals.

[00098] To further confirm the cha racteristic changes in dolichol profile, plasma samples were collected and analyzed from 6 K42E/K42E patients, 25 K42E carriers, and 16 normal individuals. The mean plasma D18/D19 ratio of K42E/K42E patients (2.75 ± 0.28, mean ± SD, n = 6) is 3.4 times higher than that of the normal individuals (0.82 ± 0.12, n = 16, P < 0.001), and 1.8 times higher than the ratio of K42E carriers (1.56 ± 0.11, n = 25, P < 0.001) (FIG. 6A). The mean plasma ratio of K42E carriers is 1.9 times higher than that of the normal individuals (P < 0.001) (FIG. 6A). The narrow distribution of the D18/D19 ratios in the carriers and normal individuals (FIG. 6A) indicates that the plasma D18/D19 ratios are not apprecia bly influenced by age, gender, or time of collection, as sample collections were untimed and without restrictions (see Materials and Methods).

[00099] ROC curve analysis shows that the plasma D18/D19 ratio discriminates K42E/K42E patients from K42E carriers with an area under curve (AUC) of 1.0 and 100% sensitivity and specificity at the cutoff level of 2.14 (FIG. 6B). The D18/D19 ratio also discriminates K42E carriers from normal individuals with an AUC of 1.0 and 100% sensitivity and specificity at the cutoff level of 1.14 (FIG. 6C).

[000100] Urinary samples were analyzed in 6 K42E/K42E patients, 30 K42E carriers, and 13 normal individuals. The mean urinary D18/D19 ratio of K42E/K42E patients (4.10 ± 0.44, mean ± SD, n = 6) is 8.7 times higher than normal individuals (0.47 ± 0. 06, n = 13, P < 0.001), and 3.2 times higher than K42E carriers (1.27 ± 0.09, n = 30, P < 0.001) (FIG. 6D). The mean urinary D18/D19 ratio of K42E carriers is 2.7 times higher than normal individuals ( P < 0.001) (FIG. 6D). As in the plasma, distribution of the D18/D19 ratios in the urine of carriers and normal individuals (FIG. 6A) is very narrow, indicating that the urinary D18/D19 ratios are not significantly influenced by age, gender, or timing of collection, as sample collections were untimed and without restrictions (see Materials and Methods). Of note, the separation of D18/D19 ratios among the three groups (K42E/K42E patients, K42E carriers, and normal individuals) in urine is higher than that it is in plasma.

[000101] ROC curve analysis indicates that the urinary D18/D19 ratio discriminates K42E/K42E patients from K42E carriers with an AUC of 1.0 and 100% sensitivity and specificity at the cutoff level of

2.46 (FIG. 6E). The D18/D19 ratio in urine also discriminates K42E carriers from normal individuals with an AUC of 1.0 and 100% sensitivity and specificity at the cutoff level of 0.85 (FIG. 6F).

[000102] FIG. 7 is a graph showing the Dol-18/Doll9 ratio in urine from K42E/K42E (Affected),

K42E (Carrier), and wt (Normal) patients indicating that urine samples can be used to assess dolichol ratios from patients. The urinary Dol-18/Doll9 ratio in urine from affected (K42E/K42E) patients are much higher than in the unaffected individuals (wt) individuals, whereas the Dol-18/Doll9 ratios of the

K42E carriers are lower than those of the patients but higher than the unaffected.

[000103] FIG. 8 is a graph showing the ratio of Dol-18:Dol-19 obtained from urine samples of 12 unaffected (WT), 15 K42E (Carrier) and 6 K42E/K42E (Affected) individuals. The urinary D18/D19 ratios of the affected patients are much higher than in the unaffected individuals, whereas the D18/D19 ratios of the K42E carriers are lower than those of the patients but higher than the unaffected.

[000104] Plasma and urinary D18/D19 ratios distinguish arRP patients carrying the K42E DHDDS mutation from arRP patients with wild-type DHDDS.

[000105] To demonstrate the predictive value of the D18/D19 ratio as a biomarker for dolichol metabolism and for the diagnosis of arRP caused by a bnormal dolichol metabolism, plasma samples from 36 arRP patients were blindly screened that had been collected at the Bascom Palmer Eye Institute since 2008. The genetic mutations of these patients had not been previously identified. Two patients (from the same family) were found to have high D18/D19 ratios of 2.66 and 3.59, above the cutoff level of 2.14 for K42E/K24E patients. Their urinary D18/D19 ratios are 3.45 and 3.93, also above the cutoff level of 2.46 for homozygous K42E mutation. Subsequent genotyping confirmed that both are homozygous for the K42E DHDDS mutation.

[000106] Dolichol profiles of the remaining 34 arRP patients were all in the normal range. The mean plasma D18/D19 ratio was 0.84 ± 0.10 (mean ± SD, n = 34), indistinguishable from that of normal individuals (0.82 ± 0.12, n = 16, P =0.60). Urinary samples were recently collected from 6 of these patients. Their mean urinary D18/D19 ratio was 0.46 ± 0.02 (n = 6), indistinguishable from that of normal individuals (0.47 ± 0.06, n = 13, P = 0.51). Together, these results demonstrate that plasma and urinary D18/D19 ratios can reliably distinguish arRP patients homozygous for the K42E DHDDS mutation from arRP patients with WT DHDDS.

[000107] Dolichol profiles of an arRP patient with compound heterozygous K42E/T206A DHDDS mutations.

[000108] A single base c.616 A>G mutation in the DHDDS-encoding gene that changes Thr206 to Ala was recently discovered in a patient with compound heterozygous K42E/T206A DHDDS mutations. Because this is the first RP patient identified to have the T206A mutation and because he is

heterozygous for the T206A allele, whether the T206A mutation was RP-causative needed to be determined. This question was addressed by dolichol analysis. The patient's urinary D18/D19 ratio was 3.39, and his plasma D18/D19 ratio was 2.59. Both are above the cutoff levels for K42E/K42E patients.

[000109] The high D18/D19 ratios could have two possible interpretations. First, the T206A mutation could be a null-equivalent loss-of-function mutation so that the high D18/D19 ratio was solely determined by the K42E allele. Alternatively, the T206A DHDDS mutant could be functionally similar to the K42E mutant and, therefore, contributing to the high D18/D19 ratios. To resolve this issue, samples from the parents were analyzed. Their urinary D18/D19 ratios were 1.12 and 1.07, both above the cutoff level of 0.85 for K42E carriers. Their plasma D18/D19 ratios were 1.55 and 1.36, respectively, also above the cutoff level of 1.13 for K42E carriers. Results from the parents strongly indicate that each parent carries a K42E-equivalent mutation. Since one parent is an obligate T206A carrier, the high D18/D19 ratios of both parents provide the first evidence that the T206A mutation affects dolichol biosynthesis in a manner similar to the K42E mutation. Thus the T206A mutation should be as RP-causative as the K42E mutation. Subsequent genotyping showed that the father carries the K42E mutation, and the mother has the T206A mutation. Five other family members also have D18/D19 ratios above the cutoff levels for carriers of DHDDS mutations (results not shown).

Genotyping identified one sibling as a carrier of the K42E mutation, and the other sibling and all three offspring as carriers of the T206A mutation.

[000110] Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and pu blications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[000111] One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

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Claims

We claim:

1. A method of treating a disease characterized by dolichol deficiency in a subject comprising administering to the subject a therapeutically effective amount of a squalene synthase inhibitor such that farnesyl-pyrophosphate pool is increased and the disease is treated.

2. The method of claim 1, in which the disease is characterized by a mutation in dehydrodolichol diphosphate synthase (DHDDS) gene.

3. The method of claim 1, in which the squalene synthase inhibitor is selected from the group consisting of zaragozic acid, lapaquistat acetate, E -27865, 2,8-dioxabicyclo[3,2,l]-octane and derivatives thereof, dicarboxylic acid, quinuclidine derivatives, 4,1-benzoxazepine, substituted morphine derivatives, phenoxypropylamines, monooxygenase synthase, and combinations thereof.

4. The method of claim 3, wherein the squalene synthase inhibitor comprises zaragozic acid.

5. The method of claim 3, wherein the squalene synthase inhibitor comprises lapaquistat acetate.

6. The method of claim 3, wherein the squalene synthase inhibitor comprises ER-27865.

7. The method of claim 3, wherein the squalene synthase inhibitor comprises

2,8-dioxabicyclo[3,2,l]-octane.

8. The method of claim 1, in which the squalene synthase inhibitor is administered at the onset of disease.

9. The method of claim 1, in which the squalene synthase inhibitor is administered prior to the onset of disease.

10. The method of claim 1, in which the squalene synthase inhibitor is administered after the onset of disease.

11. A method of preventing a disease characterized by dolichol deficiency in a subject comprising administering to the subject a therapeutically effective amount of a squalene synthase inhibitor such that farnesyl-pyrophosphate pool is increased and the disease is prevented.

12. The method of claim 11, in which the disease is characterized by a mutation in DHDDS gene.

13. The method of claim 11, in which the squalene synthase inhibitor is selected from the group consisting of zaragozic acid, lapaquistat acetate, E -27865, 2,8-dioxabicyclo[3,2,l]-octane and derivatives thereof, dicarboxylic acid, quinuclidine derivatives, 4,1-benzoxazepine, substituted morphine derivatives, phenoxypropylamines, monooxygenase synthase, and combinations thereof.

14. The method of claim 13, wherein the squalene synthase inhibitor comprises zaragozic acid.

15. The method of claim 13, wherein the squalene synthase inhibitor comprises lapaquistat acetate.

16. The method of claim 13, wherein the squalene synthase inhibitor comprises ER-27865.

17. The method of claim 13, wherein the squalene synthase inhibitor comprises

2,8-dioxabicyclo[3,2,l]-octane.

18. The method of claim 11, in which the squalene synthase inhibitor is administered at the onset of disease.

19. The method of claim 11, in which the squalene synthase inhibitor is administered prior to the onset of disease.

20. The method of claim 11, in which the squalene synthase inhibitor is administered after the onset of disease.

21. A method of ameliorating a disease characterized by dolichol deficiency in a subject comprising administering to the subject a therapeutically effective amount of a squalene synthase inhibitor such that farnesyl-pyrophosphate pool is increased and the disease is ameliorated.

22. The method of claim 21, in which the disease is characterized by a mutation in DHDDS gene.

23. The method of claim 21, in which the squalene synthase inhibitor is selected from the group consisting of zaragozic acid, lapaquistat acetate, E -27865, 2,8-dioxabicyclo[3,2,l]-octane and derivatives thereof, dicarboxylic acid, quinuclidine derivatives, 4,1-benzoxazepine, substituted morphine derivatives, phenoxypropylamines, monooxygenase synthase, and combinations thereof.

24. The method of claim 23, wherein the squalene synthase inhibitor comprises zaragozic acid.

25. The method of claim 23, wherein the squalene synthase inhibitor comprises lapaquistat acetate.

26. The method of claim 23, wherein the squalene synthase inhibitor comprises ER-27865.

27. The method of claim 23, wherein the squalene synthase inhibitor comprises

2,8-dioxabicyclo[3,2,l]-octane.

28. The method of claim 21, in which the squalene synthase inhibitor is administered at the onset of disease.

29. The method of claim 21, in which the squalene synthase inhibitor is administered prior to the onset of disease.

30. The method of claim 21, in which the squalene synthase inhibitor is administered after the onset of disease.

31. A method for identifying a subject with a dehydrodolichol diphosphate synthase (DHDDS) mutation comprising (a) obtaining a biological sample from the subject; (b) determining at least one dolichol ratio in the sample; (c) comparing at least one dolichol ratio against that of at least one control; and (d) administering a squalene synthase inhibitor if at least one of the ratios in the biological sample is higher than that of the control.

32. The method of claim 31, wherein the dolichol ratio is selected from the group consisting of dolichol 17:dolichol 19; dolichol 18:dolichol 19; dolichol 17:dolichol 20; dolichol 18:dolichol 20; dolichol 19:dolichol 20; and combinations thereof.

33. The method of claim 31, wherein the biological sample is selected from the group consisting of whole blood, plasma, serum, urine, saliva, and tissue biopsy.

34. The method of claim 33, wherein the biological sample comprises urine.

35. The method of claim 33, wherein the biological sample comprises whole blood.

36. A method for identifying a subject with a dehydrodolichol diphosphate synthase (DHDDS) mutation comprising: obtaining a biological sample from the subject;

determining at least one dolichol ratio in the sample; and

comparing the at least one dolichol ratio against that of a control,

wherein the subject is identified as having the DHDDS mutation if at least one of the ratios in the biological sample is higher than that of the control.

37. The method of claim 36, further comprising administering a squalene synthase inhibitor if at least one of the ratios in the biological sample is higher than that of the control.

38. The method of claim 36, wherein the dolichol ratio is selected from the group consisting of dolichol 17:dolichol 19; dolichol 18:dolichol 19; dolichol 17:dolichol 20; dolichol 18:dolichol 20; dolichol 19:dolichol 20 and combinations thereof.

39. The method of claim 36, wherein the dolichol ratio comprises dolichol 18:dolichol 19.

40. The method of claim 36, wherein the dolichol ratio consists of dolichol 18:dolichol 19.

41. The method of claim 36, wherein the biological sample is selected from the group consisting of whole blood, plasma, serum, urine, saliva, and tissue biopsy.

42. The method of claim 36, wherein the biological sample comprises urine.

43. The method of claim 36, wherein the biological sample comprises whole blood.

44. A method for identifying a subject having a dehydrodolichol diphosphate synthase (DHDDS) mutation, the method comprising:

determining at least one dolichol ratio in a biological sample derived from a subject; and identifying the subject as having a DHDDS mutation if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

45. The method of claim 44, wherein the dolichol ratio comprises one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20.

46. The method of claim 44, wherein the dolichol ratio comprises dolichol 18:dolichol 19.

47. The method of claim 44, wherein the dolichol ratio consists of dolichol 18:dolichol 19.

48. The method of claim 44, wherein the biological sample comprises whole blood, plasma, serum, urine, saliva, or tissue biopsy.

49. The method of claim 44, wherein the biological sample comprises urine.

50. The method of claim 44, wherein the biological sample comprises plasma.

51. The method of claim 44, wherein the dolichol ratio comprises dolichol 18:dolichol 19 and the biological sample comprises plasma or urine.

52. The method of claim 51, wherein the dolichol 18:dolichol 19 ratio is greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

53. The method of claim 44, further comprising administering a squalene synthase inhibitor if at least one of the ratios in the biological sample are higher than that of the control.

54. The method of claim 44, wherein the determining the at least one dolichol ratio is carried out by liquid chromatography-mass spectrometry (LC-MS).

55. A method for identifying a subject having a dolichol deficiency, the method comprising:

determining at least one dolichol ratio in a biological sample derived from a subject; and identifying the subject as having a dolichol deficiency if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

56. The method of claim 55, wherein the dolichol ratio comprises one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20.

57. The method of claim 55, wherein the dolichol ratio comprises dolichol 18:dolichol 19.

58. The method of claim 55, wherein the dolichol ratio consists of dolichol 18:dolichol 19.

59. The method of claim 55, wherein the biological sample comprises whole blood, plasma, serum, urine, saliva, or tissue biopsy.

60. The method of claim 55, wherein the biological sample comprises urine.

61. The method of claim 55, wherein the biological sample comprises plasma.

62. The method of claim 55, wherein the dolichol ratio comprises dolichol 18:dolichol 19 and the biological sample comprises plasma or urine.

63. The method of claim 62, wherein the dolichol 18:dolichol 19 ratio is greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

64. The method of claim 55, further comprising administering a squalene synthase inhibitor if at least one of the ratios in the biological sample are higher than that of the control.

65. The method of claim 55, wherein the determining the at least one dolichol ratio is carried out by liquid chromatography-mass spectrometry (LC-MS).

66. A kit for identifying a subject having a dehydrodolichol diphosphate synthase (DHDDS) mutation, the kit comprising:

one or more reagents for lipid extraction for determining at least one dolichol ratio in a biological sample derived from a subject; and

instructions for carrying out the determination of the at least one dolichol ratio in the biological sample and for identifying the subject as having a DHDDS mutation if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

67. The kit of claim 66, wherein the dolichol ratio comprises one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20.

68. The kit of claim 66, wherein the dolichol ratio comprises dolichol 18:dolichol 19.

69. The kit of claim 66, wherein the dolichol ratio consists of dolichol 18:dolichol 19.

70. The kit of claim 66, wherein the biological sample comprises whole blood, plasma, serum, urine, saliva, or tissue biopsy.

71. The kit of claim 66, wherein the biological sample comprises urine.

72. The kit of claim 66, wherein the biological sample comprises plasma.

73. The kit of claim 66, wherein the dolichol ratio comprises dolichol 18:dolichol 19 and the biological sample comprises plasma or urine.

74. The kit of claim 73, wherein the dolichol 18:dolichol 19 ratio is greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

75. The kit of claim 66, wherein the determining the at least one dolichol ratio is carried out by liquid chromatography-mass spectrometry (LC-MS).

76. The kit of claim 66, further comprising a sample collection kit for the biological sample.

77. A kit for identifying a subject having a dolichol deficiency, the kit comprising:

instructions for carrying out the determination of the at least one dolichol ratio in the biological sample and for identifying the subject as having a having a dolichol deficiency if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

78. The kit of claim 77, wherein the dolichol ratio comprises one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20.

79. The kit of claim 77, wherein the dolichol ratio comprises dolichol 18:dolichol 19.

80. The kit of claim 77, wherein the dolichol ratio consists of dolichol 18:dolichol 19.

81. The kit of claim 77, wherein the biological sample comprises whole blood, plasma, serum, urine, saliva, or tissue biopsy.

82. The kit of claim 77, wherein the biological sample comprises urine.

83. The kit of claim 77, wherein the biological sample comprises plasma.

84. The kit of claim 77, wherein the dolichol ratio comprises dolichol 18:dolichol 19 and the biological sample comprises plasma or urine.

85. The kit of claim 84, wherein the dolichol 18:dolichol 19 ratio is greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

86. The kit of claim 77, wherein the determining the at least one dolichol ratio is carried out by liquid chromatography-mass spectrometry (LC-MS).

87. The kit of claim 77, further comprising a sample collection kit for the biological sample.

88. A method for identifying a subject that has altered dolichol biosynthesis, the method comprising:

determining at least one dolichol ratio in a biological sample derived from a subject; and identifying the subject as having altered dolichol biosynthesis if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

89. The method of claim 88, wherein the dolichol ratio comprises one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20.

90. The method of claim 88, wherein the dolichol ratio comprises dolichol 18:dolichol 19.

91. The method of claim 88, wherein the dolichol ratio consists of dolichol 18:dolichol 19.

92. The method of claim 88, wherein the biological sample comprises whole blood, plasma, serum, urine, saliva, or tissue biopsy.

93. The method of claim 88, wherein the biological sample comprises urine.

94. The method of claim 88, wherein the biological sample comprises plasma.

95. The method of claim 88, wherein the dolichol ratio comprises dolichol 18:dolichol 19 and the biological sample comprises plasma or urine.

96. The method of claim 95, wherein the dolichol 18:dolichol 19 ratio is greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

97. The method of claim 88, further comprising administering a squalene synthase inhibitor if at least one of the ratios in the biological sample are higher than that of the control.

98. The method of claim 88, wherein the determining the at least one dolichol ratio is carried out by liquid chromatography-mass spectrometry (LC-MS).

99. A kit for identifying a subject that has altered dolichol biosynthesis, the kit comprising:

instructions for carrying out the determination of the at least one dolichol ratio in the biological sample and for identifying the subject as having altered dolichol biosynthesis if the at least one dolichol ratio is higher in the biological sample derived from the subject as compared to a reference control.

100. The kit of claim 99, wherein the dolichol ratio comprises one or a combination of dolichol 17:dolichol 19, dolichol 18:dolichol 19, dolichol 17:dolichol 20, dolichol 18:dolichol 20, and dolichol 19:dolichol 20.

101. The kit of claim 99, wherein the dolichol ratio comprises dolichol 18:dolichol 19.

102. The kit of claim 99, wherein the dolichol ratio consists of dolichol 18:dolichol 19.

103. The kit of claim 99, wherein the biological sample comprises whole blood, plasma, serum, urine, saliva, or tissue biopsy.

104. The kit of claim 99, wherein the biological sample comprises urine.

105. The kit of claim 99, wherein the biological sample comprises plasma.

106. The kit of claim 99, wherein the dolichol ratio comprises dolichol 18:dolichol 19 and the biological sample comprises plasma or urine.

107. The kit of claim 106, wherein the dolichol 18:dolichol 19 ratio is greater than about 2-fold higher in the biological sample derived from the subject as compared to the reference control.

108. The kit of claim 99, wherein the determining the at least one dolichol ratio is carried out by liquid chromatography-mass spectrometry (LC-MS).

109. The kit of claim 99, further comprising a sample collection kit for the biological sample.