Method of Diagnosis of Chronic Kidney Disease with Pharmacogenomics Support

Abstract

A method for determining a subject's risk for developing chronic kidney disease (CKD) combined with determining a subject's pharmacogenomic (PGX) profile is described. The method involves obtaining a sample of genetic material from the subject. The genetic material is amplified using primers specific for the genes underlying CKD and PGX. The DNA sequence of the amplified genetic material is determined and compared with the human reference genome sequence. One or more DNA sequence alterations in the amplified genetic material not present in the human reference genome sequence determines the PGX profile of the subject and may indicate that the subject is at risk for developing CKD.

Claims

1. A method for determining a subject's PGX profile and risk for developing CKD, the method comprising the steps of: (a) obtaining a sample of genetic material from the subject; (b) amplifying the genetic material using two or more primers specific for the 334 genes underlying CKD and 40 genes underlying PGX; (c) determining the DNA sequence of the amplified genetic material of step (b); and (d) comparing the DNA sequence of the amplified genetic material with a DNA sequence from a human reference genome; (e) wherein one or more DNA sequence alterations in the amplified genetic material indicates the subject's PGX profile and that the subject has a risk for developing CKD.

2. The method of claim 1, wherein the DNA sequence alteration is a variant.

3. The method of claim 1, wherein the DNA sequence alteration is a mutation.

4. The method of claim 1, wherein the DNA sequence alteration is a polymorphism.

5. The method of claim 1, wherein the step of amplification of the sample of genetic material comprises amplification of 374 total genes.

6. The method of claim 1, wherein the genes underlying CKD and PGX comprise the gene lists from Table 1 and Table 2.

7. The method of claim 1, wherein the subject's risk for developing CKD and their PGX profile are determined within 48 hours of receipt of the sample from the subject.

8. The method of claim 1, wherein the subject's risk for developing CKD and their PGX profile are determined within 5 days of receipt of the sample of from the subject.

9. A method for diagnosing CKD and determining the PGX profile of a subject, the method comprising the steps of: (a) obtaining a sample of genetic material from the subject; (b) amplifying the genetic material using primers specific for the genes underlying CKD and PGX; (c) determining the DNA sequence of the amplified genetic material of step (b); and (d) comparing the DNA sequence of the amplified genetic material with a DNA sequence from the human reference genome sequence; (e) wherein one or more DNA sequence alterations in the amplified genetic material not present in the DNA sequence from the human reference genome sequence determines the PGX profile of the subject and may indicate that the subject has CKD.

10. The method of claim 9, wherein the DNA sequence alteration is a variant.

11. The method of claim 9, wherein the DNA sequence alteration is a mutation.

12. The method of claim 9, wherein the DNA sequence alteration is a polymorphism.

13. The method of claim 9, wherein the step of amplification of the sample of genetic material comprises amplification of 374 genes.

14. The method of claim 9, wherein the genes underlying CKD and PGX comprise the gene lists from Table 1 and Table 2.

15. The method of claim 9, wherein the genetic results are determined within 48 hours of receipt of the sample of from the subject.

16. The method of claim 9, wherein the genetic results are determined within 5 days of receipt of the sample of from the subject.

17. A method for determining a subject's risk for being a genetic carrier for CKD or PGX haplotypes, the method comprising the steps of: (a) obtaining a sample of genetic material from the subject; (b) amplifying the genetic material using primers specific for the genes underlying CKD and PGX; (c) determining the DNA sequence of the amplified genetic material of step (b); and (d) comparing the DNA sequence of the amplified genetic material with a DNA sequence from the human reference genome sequence; (e) wherein one or more DNA sequence alterations in the amplified genetic material not present in the human reference genome sequence determines the subject's PGX profile and may indicate that the subject is a genetic carrier for CKD.

18. The method of claim 17, wherein the DNA sequence alteration is a variant.

19. The method of claim 17, wherein the DNA sequence alteration is a mutation.

20. The method of claim 17, wherein the DNA sequence alteration is a polymorphism.

Description

DESCRIPTION

[0014] According to one embodiment of the present invention, there is provided a method for rapid detection of variants, mutations, structural variants and polymorphisms associated with CKD and PGX. The time for diagnosing or determining the patient's risk of developing CKD and their PGX profile using the method of the present invention can be as early as 48 hours after receipt of the patient's sample. The method of the present invention can also be used to diagnose CKD and determine their PGX profile within 5 days after receipt of the patient's sample. The method involves analysis of 334 CKD genes and 40 PGX genes, referred to herein as the “genes of interest.”

[0015] As used in this disclosure, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising,” “comprises” and “comprised” are not intended to exclude other additives, components, integers or steps. Thus, throughout this specification, unless the context requires otherwise, the words “comprise,” “comprising” and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of “including, but not limited to.”

[0016] As used in this disclosure, except where the context requires otherwise, the method steps are not intended to be limiting nor are they intended to indicate that each step is essential to the method or that each step must occur in the order disclosed.

[0017] As used herein, “sample” refers to any sample that can be from or derived a human patient, e.g., bodily fluids (blood, saliva, urine etc.), biopsy, tissue, and/or waste from the patient. Thus, tissue biopsies, stool, sputum, saliva, blood, lymph, tears, sweat, urine, vaginal secretions, or the like can be used in the method, as can essentially any tissue of interest that contains the appropriate nucleic acids. The sample may be in a form taken directly from the patient. Preferably, the sample may be at least partially purified to remove at least some non-nucleic acid material.

[0018] The term “DNA sequence” as used herein refers to chromosomal sequence as well as to cDNA sequence.

[0019] The term “amplifying” in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced. Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase based amplification (e.g., by transcription) methods.

[0020] An “amplicon” is an amplified nucleic acid, e.g., a nucleic acid that is produced by amplifying a template nucleic acid by any available amplification method (e.g., PCR, LCR, transcription, or the like).

[0021] A “gene” is one or more sequence(s) of nucleotides in a genome that together encode one or more expressed molecules, e.g., an RNA. The gene can include coding sequences that are transcribed into RNA which may then be translated into a polypeptide sequence, and can include associated structural or regulatory sequences that aid in replication or expression of the gene.

[0022] A “set” or “pool” of primers or amplicons refers to a collection or group of primers or amplicons, or the data derived therefrom, used for a common purpose, e.g., identifying an individual with a specified genotype (e.g., risk of developing CKD). Frequently, data corresponding to the primers or amplicons, or derived from their use, is stored in an electronic medium.

[0023] A “structural variant” refers to a variation of a DNA sequence such as, for example, a deletion, duplication, insertion, translocation or inversion.

[0024] A set of oligonucleotide sequences, or primers, have been identified that facilitate the rapid identification of variants, mutations, polymorphisms, and other structural variants associated with CKD and PGX. This set of oligonucleotides detects sequences that are indicative of whether an individual is a carrier for, or has CKD, and their type of PGX profile.

[0025] The primers are used to amplify certain genes underlying CKD and PGX. This includes targeting all exons of the genes, plus 5 base pairs of flanking intronic DNA, plus deep intronic and promoter regions and the 5 prime and 3 prime untranslated regions of the genes, and any structural variants known to be associated with CKD and PGX.

[0026] The DNA used in the method of the invention can be extracted from any source, such as, for example, whole blood, samples from a buccal swab, or it can be from previously extracted genomic DNA samples. Preferably, the DNA is extracted from whole blood using known techniques. Prior to analysis, extracted DNA can be stored for one month at room temperature or 2-8° C. For longer storage of up to 2 years, DNA can be stored frozen at <−20° C. to minimize the degradation of nucleic acid.

[0027] After extraction, the DNA sample is measured and assessed for purity. 30 ng of genomic DNA, at a minimum concentration of 1.7 ng/μL, is preferable for analysis, but as little as 1 ng is sufficient. The concentration of purified DNA should preferably be adjusted to between 2 ng/μL and 25 ng/μL prior to analysis. Optimal DNA purity is an absorbance ratio (A.sub.260/A.sub.280) of 1.80 or greater (typical range is 1.60 to 2.00).

[0028] The extracted sample DNA is then amplified. A well-known amplification method, for example, is the polymerase chain reaction (PCR). In PCR, a characteristic piece of the particular nucleotide sequence of interest is amplified with specific primers. If the primer pair finds its target site, a sequence of the genetic material undergoes a million-fold proliferation.

[0029] During the PCR process, the DNA generated is used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. PCR can amplify a single or few copies of a piece of DNA by several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations, as known by one of skill in the art.

[0030] In the method described herein, unique primer pool aliquots are used to amplify the region of interest from the sample. The primer pool aliquots contain forward and reverse primer pairs directed towards one or more of the 334 CKD genes or the 40 PGX genes, as listed in Table 1 and Table 2.

TABLE-US-00001 TABLE 1 CKD Genes Gene Cytogenetic OMIM Gene MIM Name Location Number Disease Name ACE 17q23 106180 Renal tubular dysgenesis ACTN4 19q13 604638 Focal Segmental Glomerulosclerosis 1 ADAMTS13 9q34.2 604134 Thrombotic thrombocytopenic purpura, familial ADCK4 Nephrotic syndrome, type 9 ADCY10 605205 Hypercalciuria, absorptive, susceptibility to AGT 1q42-43 106150 Renal tubular dysgenesis AGTR1 3q24 106165 Renal tubular dysgenesis AGXT 2q37.3 604285 Hyperoxaluria, primary, type 1 AHI1 6q23.3 608894 Joubert Syndrome 3 ALMS1 2p13.1 606844 Alstrom syndrome ANKS6 9q22.33 615370 Nephronophthisis 16 APOA1 11q23.3 107680 Amyloidosis, familial visceral, 3 or more types APOE APOL1 APRT 16q24.3 102600 Adenine phosphoribosyltransferase deficiency AQP2 12q12-q13 107777 Diabetes insipidus, nephrogenic ARHGDIA 17q25.3 601925 Nephrotic syndrome type 8 ARL13B 3q111 608922 Nephronophtisis, Joubert syndrome 8 ARL6 3q11.2 608845 Bardet-biedl syndrome 3 ATP6V0A4 7q34 605239 Renal tubular acidosis, distal AVPR2 Xq28 300538 Nephrogenic syndrome of inappropriate diuresis Diabetes insipidus, nephrogenic B9D2 19q13.2 611951 Meckel syndrome type 10 BBS1 11q13.2 209901 Bardet-biedl syndrome 1 BBS10 12q21.2 610148 Bardet-biedl syndrome 10 BBS12 4q27 610683 Bardet-biedl syndrome 12 BBS2 16q12.2 606151 Bardet-biedl syndrome 2 BBS4 15q24.1 600374 Bardet-Biedl Syndrome 4 BBS5 2q31.1 603650 Bardet-Biedl Syndrome 5 BBS7 4q27 607590 Bardet-biedl syndrome 7 BBS9 7p14.3 607968 Bardet-biedl syndrome 9 BMP4 14q22-23 112262 Microphthalmia, syndromic 6 BSND 1p32.1 606412 Bartter Syndrome, Type 4a, neonatal CA2 8q21.2 611492 Osteopetrosis with renal tubular acidosis CASR 3q13 601199 Neonatal severe primary hyperparathyroidism, Hypocalcemia with 7mmune syndrome CC2D2A 4p15.32 612013 Joubert syndrome with hepatic defect, Joubert syndrome with oculorenal defect, Meckel Syndrome 6, Jouber syndrome 9, COACH syndrome CD151 11p15.5 602243 Nephropathy with pretibial epidermolysis bullosa and deafness CD2AP CEP164 11q23.3 614848 Senior-Loken syndrome, Nephronopthisis 15 CEP290 12q21.32 610142 Joubert syndrome with oculorenal defect, Bardet-biedl syndrome 14, Joubert Syndrome 5, Meckel syndrome 4, Senior-Loken Syndrome 6 CEP41 7q32.2 610523 Joubert's syndrome type 15 CFH 1q31.3 134370 Complement factor h deficiency CFHR5 1q31.3 608593 Nephropathy due to CFHR5 deficiency CFI 4q25 217030 Complement factor 1 deficiency CHD7 8q12.2 608892 CHARGE syndrome CLCN5 Xp11.23 300008 Dent disease nephrolithiasis type 1 hypophosphatemic Rickets CLCNKB 1p36.13 602023 Bartter syndrome, type 3 and type 4b CLDN16 3q28 603959 Hypomagnesemia 3, renal CLDN19 1p34.2 610036 Familial primary hypomagnesemia with hypercalciuria and nephrocalcinosis with severe ocular involvementHypomagnesemia 5, renal, with ocular involvement COL4A3 2q36-q37 120070 Alport syndrome COL4A4 2q35-q37 120131 Alport syndrome; nephrotic syndrome (FSGS 1) COL4A5 Xq22.3 303630 Alport syndrome COL4A6 303631 COQ2 4q21.23 609825 Coenzyme q10 deficiency COQ6 14q24.3 614647 Coenzyme Q10 deficiency, primary, 6 CREBBP 16p133 600140 Rubinstein Taybi syndrome type 1 CSPP1 8q13.1-q13.2 611654 Meckel syndrome, Joubert syndrome 21 CTNS 17p13 606272 Cystinosis, nephropathic cystinosis, late-onset juvenile or adolescent nephropathic CUBN 10p13 602997 Megaloblastic anemia 1-finnish type CUL3 2q36.2 603136 Pseudohypoaldosteronism, type 2e CYP11B1 8q24.3 610613 Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency DGKE 17q22 601440 Nephrotic syndrome type 7 DHCR7 11q13.4 602858 Smith-Lemli-Opitz Syndrome DSTYK 1q321 612666 Congenital Anomalies of the Kidney and the Urinary Tract EGF 4q25 131530 Hypomagnesemia 4, renal EYA1 8q13.3 601653 Branchio-oto-renal syndrome 1 FAN1 15q13.2-q13.3 613534 Interstitial nephritis, karyomegalic FGA 4q31.3 134820 Amyloidosis, familial visceral, hereditary renal FGF20 8p22 605558 Renal hypodysplasiaaplasia 2 FGF23 12p13.3 605380 Hypophosphatemic Rickets, autosomal dominant Tumoral Calcinosis, hyperphosphatemic, familial FGFR1 8p12 136350 Encephalocraniocutaneous Lipomatosis, Kallmann syndrome 2, kal2 FN1 2q34 135600 Glomerulopathy with fibronectin deposits 2 FRAS1 4q21.21 607830 Fraser syndrome FREM1 9p22.3 608944 Bifid nose with or without anorectal and renal anomalies FXYD2 11q23.3 601814 Hypomagnesemia 2, renal GDNF 5p131-p12 600837 Congenital Anomalies of the Kidney and the Urinary Tract GLA Xq22.1 300644 Fabry Disease GLIS2 16p13.3 608539 Nephronophthisis 7 GPC3 Xq26.2 300037 Simpson golabi behmel syndrome type 1 GRHPR 9p13.2 604296 Hyperoxaluria, primary, type 2 GSN 9q33.2 137350 Amyloidosis, finnish type cerebral amyloid angiopathy, gsn-related HNF1B 17q12 189907 Renal Cysts and Diabetes Syndrome HSD11B2 16q22.1 614232 Apparent mineralocorticoid excess INF2 14q32.33 610982 Focal Segmental Glomerulosclerosis 5 Charcot-Marie-Tooth disease E INPP5E 9q34.3 613037 Joubert syndrome with hepatic defectJoubert Syndrome 1 INVS 9q31.1 243305 Senior-Loken syndrome, Nephronophthisis 2, infantile IQCB1 3q13.33; 3q21.1 609237 Senior-loken syndrome 5 JAG1 20p12.2 601920 Alagille syndrome type 1 KANK1 9p24.3 607704 Nephroticsyndrome KCNJ10 1q23.2 602208 Seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (sesame syndrome) KCNJ5 11q243 600734 Hyperaldosteronism, familial, type 3 KIF7 15q26.1 611254 Acrocallosal syndrome KLHL3 5q31.2 605775 Pseudohypoaldosteronism, type 2d LAMB2 3p21.31 150325 Nephrotic syndrome, type 5, with or without ocular abnormalities Pierson Syndrome LMX1B 9q33.3 602575 Nail-patella syndrome LRP4 11p11.2 604270 Cenani-lenz syndactyly syndrome LYZ 12q15 153450 Amyloidosis, familial visceral, renal MAGI2 606382 191170 MEFV 16p13.3 608107 Familial Mediterranean fever MKKS 20p12.2 604896 Bardet-biedl syndrome 6, McKusick- Kaufman syndrome MKS1 17q22 609883 Meckel syndrome 1, Bardet-Biedl syndrome 13 MMACHC 1p34.1 609831 Methylmalonic aciduria and homocystinuria, cblc type MUC1 1q22 158340 Medullary cystic kidney disease 1 MYH9 22q13.1 160775 Fechtner syndrome; Epstein Syndrome MYO1E 15q21-q22 601479 Glomerulosclerosis, focal segmental, 6 NEK8 17q11.1 609799 Nephronophthisis 9 Renal-heaptic- pancreatic dysplasia 2 NLRP3 1q44 606416 Muckle-wells syndrome Familial cold- induced inflammatory syndrome 1 NOTCH2 1p12 600275 Acroosteolysis dominant type, Alagille syndrome 2, Hajdu-Cheney Syndrome NPHP1 2q13 607100 Bardet-Biedl syndrome, Nephronophthisis 1 (juvenile), Senior- Loken syndrome, Joubert syndrome 4 NPHP3 3q22.1 608002 Senior-Loken syndrome, Nephronophthisis 3 (adolescent), Meckel syndrome 7, Renal-hepatic- pancreatic dysplasia NPHP4 1p36 607215 Nephronophthisis 4 Senior-Loken syndrome 4 NPHS1 19q13.12 602716 Congenital nephrotic syndrome, Finnish type NPHS2 1q25.2 604766 Nephrotic syndrome type 2 NR3C2 4q31.1 600983 Pseudohypoaldosteronism Type 1, autosomal dominant Hypertension, Early-Onset, Autosomal Dominant, With Exacerbation in Pregnancy OCRL Xq25 300535 Dent disease 2 Lowe syndrome OFD1 Xp22.2 300170 Joubert syndrome type 10, Orofaciodigital syndrome I, Golabi- Behmel syndrome, type 2 PAX2 10q24.31 167409 Renal coloboma syndrome, FSGS 7, Papillorenal Syndrome, PAPRS PDSS2 6q21 610564 Coenzyme q10 deficiency, primary, 3 PHEX Xp22.2-p22.1 300550 Hypophosphatemic rickets PKD1 16p13.3 601313 Polycystic kidney disease 1 PKD2 4q22.1 173910 Polycystic kidney disease 2 PKHD1 6p12.3-p12.2 606702 autosomal recessive polycystic kidney disease, Polycystic and hepatic disease PLCE1 10q23.3 608414 Nephrotic syndrome type 3 PTPRO 12p13.3-p13.2; 600579 Nephrotic syndrome, type 6 12p13-p12 REN 1q32.1 179820 Renal tubular dysgenesis hyperuricemic nephropathy, familial juvenile 2 RET 10q11.21 164761 Renal agenesis, bilateral ROBO2 3p12.3 602431 Vesicoureteral reflux 2 RPGRIP1L 16q12.2 610937 Meckel syndrome 5, Joubert syndrome 7, COACH syndrome SALL1 16q12.1 602218 Townes-Brocks Branchiootorenal-like Syndrome SALL4 20q13.2 607343 Acro-renal-ocular syndrome, Duane- Radial Ray Syndrome IVIC Syndrome SCARB2 4q21.1 602257 Epilepsy, Progressive Myoclonic, 4 with or without Renal Failure SCNN1A 12p13.31 600228 Pseudohypoaldosteronism, Type I SCNN1B 16p12.2 600760 Liddle syndrome SCNN1G 16p12 600761 Pseudohypoaldosteronism, type 1 Liddle Syndrome SDCCAG8 1q43 613524 Senior loken syndrome type 7, Bardet- Biedl Syndrome 16 SEMA3E 7q21.11 608166 Charge syndrome SIX1 14q23.1 601205 Branchio-oto-renal syndrome SIX2 2p21 604994 Renal hypodysplasia SIX5 19q13.32 600963 Branchio-oto-renal syndrome 2 SLC12A1 15q21.1 600839 Bartter syndrome, type 1, antenatal SLC12A3 16q13 600968 Gitelman syndrome SLC22A12 11q13.1 607096 Hypouricemia, renal, 1, rhuc1 SLC34A1 5q35.3 182309 Fanconi renotubular syndrome 2 hypercalcemia, infantile, 2 nephrolithiasis, osteoporosis, hypophosphatemic, 1 SLC34A3 9q34.3 609826 Hypophosphatemic rickets with hypercalciuria, hereditary SLC3A1 2p21 104614 Cystinuria SLC4A1 17q21.31 109270 Renal tubular acidosis, distal SLC4A4 4q13.3 603345 Renal tubular acidosis, proximal, with ocular abnormalities and mental retardation SLC5A2 16p11.2 182381 Renal glucosuria SLC7A9 19q13.1 604144 Cystinuria SLC9A3R1 17q25.1 604990 Nephrolithiasis, osteoporosis, hypophosphatemic, 2 SLIT2 4p152 603746 Congenital Anomalies of the Kidney and the Urinary Tract SMARCAL1 2q35 606622 Schimke 12mmune-osseous dysplasiaSchimke's immunoosseous dystrophy TCTN1 TMEM216 11q12.2 613277 Joubert syndrome 2, Meckel syndrome TMEM237 2q33.1 614423 Joubert syndrome type 14 Nephronophtisis 11, Meckel Syndrome TMEM67 8q22.1 609884 3, Joubert syndrome 6, COACH syndrome TNFRSF1A 12p1331 191190 Autosomal dominant periodic fever syndrome TRAP1 16p133 606219 Congenital Anomalies of the Kidney and the Urinary Tract TRIM32 9q33.1 602290 Bardet-biedl syndrome 11 TRPC6 11q22.1 603652 Focal Segmental Glomerulosclerosis 2 TRPM6 9q21.13 607009 Hypomagnesemia 1, intestinal TSC1 9q34 605284 Tuberous Sclerosis-1 TSC2 16p13.3 191092 Tuberous sclerosis-2 TTC21B 2q24.3 612014 Nephronophthisis 12 TTC8 14q31.3 608132 Bardet-Biedl Syndrome 8 TTR 18q12.1 176300 Amyloidosis, hereditary, transthyretin- related UMOD 16p12.3 191845 Medullary cystic kidney disease 2 hyperuricemic nephropathy glomerulocystic kidney disease UPK3A 22q1331 611559 Renal hypodysplasia, urogenital dysplasia VHL 3p25.3 608537 Von Hippel-Lindau Syndrome VIPAS39 14q24.3 613401 Arthrogryposis, renal dysfunction, and cholestasis 2; arcs2 VPS33B 15q26.1 608552 Arthrogryposis, renal dysfunction, and cholestasis 1; arcs1 WDPCP 2p15 613580 Bardet-Biedl syndrome WDR19 4p14 608151 Senior-Loken syndrome, Short-rib thoracic dysplasia 5, Nephronophthisis type 13, Cranioectodermal dysplasia WDR35 2p24.1 613602 Short rib-polydactyly syndrome, Verma-Naumoff type, Short rib- polydactyly syndrome (Jeune's syndrome) type 7, Cranioectodermal dysplasia type 2 WNK1 12p13.33 605232 Pseudohypoaldosteronism, type 2c WNK4 17q21.2 601844 Pseudohypoaldosteronism, type 2b WT1 11p13 607102 Denys-Drash syndrome, Frasier syndrome, Nephrotic syndrome type 4, Wilms tumor type 1 XDH 2p23.1 607633 Xanthinuria, type i XPNPEP3 22q13.2 613553 Nephronophthisis-like nephropathy 1 ZNF423 16q12.1 604557 Nephronophthisis-14 Joubert syndrome 16 ABCG2 Susceptibility to Gout ALG1 Congenital disorder of glycosylation, type Ik ALG8 11q14.1 608103 Polycystic liver disease 3 with or without kidney cysts ANLN 7p14.2 616027 Focal Segmental Glomerulosclerosis 8 ANOS1 Xp22.31 300836 Hypogonadotropic hypogonadism 1 with or without anosmia (Kallmann syndrome 1) AP2S1 19q13.32 602242 Hypocalciuric hypercalcemia, familial type 3 APOPT1 14q32.33 616003 Mitochondrial Complex 4 deficiency ARHGAP24 610586 ARMC9 Joubert syndrome 30 ATP6V1B1 2p13.3 192132 Renal tubular acidosis with deafness ATP7B 13q14.3 606882 Wilson Disease ATXN10 Spinocerebellar ataxia 10 B2M 15q21.1 109700 Amyloidosis, familial visceral B9D1 17p11.2 614144 Meckel syndromeMeckel-gruber syndrome type 9 BBIP1 10q25.2 613605 Bardet-biedl syndrome 18 BCS1L 2q35 603647 Mitochondrial complex 3 deficiency, nuclear type 1 BICC1 BMP7 112267 CAKUT C2CD3 Orofaciodigital syndrome XIV C3 19p13.3 120700 Complement component 3 deficiency CACNA1H CCDC28B Bardet-Biedl syndrome 1, modifier of CD46 CDC5L 6p21 602868 Congenital Anomalies of the Kidney and the Urinary Tract CDKN1C 11p15.5 600856 Beckwith-wiedemann syndrome IMAGE syndrome CEP104 1p36.32 616690 Joubert syndrome 25 CEP83 12q22 615847 Nephronophthisis 18 CFAP418 Bardet-Biedl syndrome 21 CFB CFHR1 CFHR3 CHD1L CHRM3 1q43 118494 Prune belly syndrome, Eagle-barrett syndrome CHRNA3 Bladder dysfunction, autonomic, with impaired pupillary reflex and secondary CAKUT CLCN2 CLCNKA 1p36.13 602024 Bartter's Syndrome Type 4b, Neonatal CLDN10 CNNM2 10q24.32 607803 Hypomagnesemia type 6 Hypomagnesemia, seizures, and mental retardation COL4A1 13q34 120130 Hereditary angiopathy with nephropathy, aneurysms and Msucle cramps (HANAC) COQ8B 19q13.1 615567 Nephrotic syndrome, type 9 COX10 17p12 602125 Mitochondrial Complex 4 deficiency COX14 12q13.12 614478 Mitochondrial Complex 4 deficiency COX20 1q44 614698 Mitochondrial Complex 4 deficiency COX6B1 19p13.12 124089 Mitochondrial Complex 4 deficiency COX8A 11q13.1 123870 Mitochondrial Complex 4 deficiency CPLANE1 Joubert CPT2 1p32 600650 Lethal neonatal carnitine palmitoyltransferase 2 deficiency CRB2 9q33.3 609720 FSGS 9 Ventriculomegaly with cystic kidney disease CRKL Congenital Anomalies of the Kidney BMP4 and Urinary Tract (CAKUT) CTU2 CYP24A1 20q13.2 126065 Hypercalcemia infantile, 1 CYP27B1 DACH1 DCDC2 6p22.3 605755 Nephronophthisis 19 DIS3L2 2q37.1 614184 Perlman's syndrome (nephroblastomatosis, gigantism) DLC1 604258 603089 DLG1 4q22.1 600980 CAKUT DMP1 Hypophosphataemic rickets EHHADH 3q27.2 607037 Fanconi renotubular syndrome 3 EIF2AK3 2p12 604032 Wolcott-Rallison Syndrome EMP2 16p13.13 602334 Nephrotic syndrome type 10 ENPP1 6q23.2 173335 Hypophosphataemicrickets ETFA 15q23-q25 608053 Glutaric aciduria 2a ETFB 19q13.3 130410 Glutaric acidemia 2b ETFDH 4q32.1 231675 Glutaric acidemia 2c FAH 15q25.1 613871 tyrosinemia, type 1 FAT1 600976 604373 FOXC2 16q24.1 602402 Lymphedema-distichiasis syndrome with renal disease and diabetes mellitus FOXP1 605515 CAKUT FREM2 13q13.3 608945 Fraser syndrome FRTS G6PC 17q21.31 613742 Glycogen storage disease la GALNT3 2q24.3 601756 Tumoral calcinosis, hyperphosphatemic, familial, hftc GANAB Polycystic kidney disease 3 GATA3 10p14 131320 Hypoparathyroidism, deafness, renal disease syndrome GATM Fanconi renotubular syndrome 1 GLI3 6p14.1 165240 Pallister-Hall syndrome GLIS3 9p24.2 610192 Diabetes Mellitus, Neonatal, With Congenital Hypothyroidism GNA11 19p13.3 139313 Hypocalciuric hypercalcemia, type 2 GREM1 GRIP1 12q14.3 604597 Fraser syndrome HNF4A 20q13.12 600281 Renal cysts and diabetes syndrome Hyperinsulinism due to HNF4A deficiency; Fanconi renotubular syndrome 4, with maturity-onset diabetes of the young HOGA1 10q24.2 613597 Primary hyperoxaluria type 3 HOXA13 7p15.2 142959 Hand-foot-uterus syndrome HOXD13 2q311 142989 Vacterl association with hydrocephalus HPRT1 Xq26.2-q26.3 308000 Kelley-Seegmiller Syndrome Lesch- Nyhan Syndrome HPSE2 10q24.2 613469 Ochoa syndromeUrofacial syndrome 1 IFT122 3q21.3-q22.1 606045 Cranioectodermal dysplasia type 1 IFT140 16p13.3 614620 Short rib-polydactyly syndrome (Jeune's syndrome) type 9 IFT172 2p23.3 607386 Bardet-Biedl syndrome, Short rib- polydactyly syndrome (Jeune's syndrome) type 10 IFT27 22q12.3 615870 Bardet-Biedl syndrome 19 IFT43 14q24.3 614068 Cranioectodermal dysplasia type 3 IFT74 Bardet-Biedl IKBKAP 9q31 603722 Familial dysautonomia, Hereditary sensory and autonomic neuropathy type 3, hsan3 ITGA3 17q21.33 605025 Interstitial lung disease, nephrotic syndrome, and epidermolysis bullosa, congenital ITGA8 10p13 604063 Renal hypodysplasia-aplasia 1 ITGB4 17q25.1 147557 Epidermolysis bullosa, junctional, with pyloric atresia KCNJ1 11q24.3 600359 Bartter's syndrome, type 2 KCNQ1OT1 Beckwith-Wiedemann syndrome KCTD1 18q11.2 613420 Scalp-Ear-Nipple Syndrome KIF12 CAKUT KIF14 1q32.1 611279 Meckel syndrome 12 LCAT 16q22.1 606967 Norum disease LMNA 1q22 150330 Atypical Werner syndrome Laminopathy type Decaudain- Vigouroux LMNA-related cardiocutaneous progeria syndrome, Restrictive Dermopathy, Lethal LRIG2 1p13.1 608869 Ochoa syndrome, Urofacial Syndrome 2 LZTFL1 3p21.31 606568 Bardet Biedl syndrome type 17 MAFB 20q12 608968 Multicentric carpo-tarsal osteolysis with or without nephropathy MAGED2 Xp11.21 300470 Bartter syndrome, type 5, antenatal, transient MMUT 6p12.3 609058 Vitamin B12-unresponsive methylmalonic acidemia type mutO MOCOS Xanthinuria MYCN 2p24.3 164840 Feingold syndrome NEK1 4q33 604588 Short rib-polydactyly syndrome (Jeune's syndrome) type 6 NSD1 5q35 606681 Sotos syndrome 1; Beckwith- Wiedemann syndrome, BWS NUP107 12q15 607617 Nephrotic syndrome, type 11 NUP205 7q33 614352 Nephrotic syndrome, type 13 NUP93 16q13 614351 Nephrotic Syndrome, Type 12 PBX1 176310 Congenital anomalies of kidney and urinary tract syndrome with or without hearing loss, abnormal ears, or developmental delay PLCG2 PMM2 16p13.2 601785 Congenital disorder of glycosylation, type Ia PTHB1 Bardet-Biedl syndrome 9 SARS2 19q13.2 612804 Hyperuricemia, pulmonary hypertension, renal failure and alkalosis SGPL1 603729 Nephrotic syndrome, type 14 SLC26A1 Nephrolithiasis, calcium oxalate SLC2A2 3q26.2 138160 Fanconi-Bickel syndrome SLC2A9 4p16.1 606142 Hypouricemia, renal, 2 SLC36A2 5q33.1 608331 Hyperglycinuria SLC41A1 SLC5A1 Renal Glucosuria SLC6A19 5p15.33 608893 Hartnup Disorder Hyperglycinuria SLC6A20 3p21.31 605616 Hyperglycinuria SLC7A7 14q11.2 603593 Lysinuric protein intolerance SMC1A Xp11.22 300040 Cornelia de Lange syndrome SOX17 8q11.23 610928 Vesicoureteral Reflux 3 SOX18 20q13.33 601618 Hypotrichosis-lymphedema- telangiectasia-renal defect syndrome SRGAP1 606523 TBX18 6q14.3 604613 Congenital hydronephrosisCongenital anomalies of Kidney and Urinary Tract 2 TCTN2 12q24.31 613846 Meckel-gruber syndrome type 8, Joubert syndrome 24 TCTN3 10q24.1 613847 Joubert syndrome 18, Orofaciodigital syndrome 4 TFAP2A 6p24.3 107580 Branchiooculofacial Syndrome THBD TMEM107 TMEM138 qq112.2 614459 Joubert syndrome 16 TNXB 6p21.33-p21.32 600985 Vesicoureteral Reflux 8 Ehlers-Danlos syndrome due to tenascin-X deficiency TP63 3q28 603273 EEC syndrome, Ectrodactyly, ectodermal dysplasia, and cleft lip, palate syndrome 3 VDR 601769 WDR73 15q25.2 616144 Galloway-Mowat syndrome WFS1 4p16.1 606201 Wolfram syndrome type 1 WNT4 1p36.12 603490 SERKAL syndrome Mullerian aplasia and hyperandrogenism XPO5 607845 ACTG1 Congenital anomalies of kidney and urinary tract ANKFY1 607927 ATP6B1 Distal renal tubular acidosis 2 with progressive sensorineural hearing loss FAM20A 17q24.2 611062 Amelogenesis imperfecta- nephrocalcinosis syndromeAmelogenesis imperfecta type IG (enamel-renal syndrome) FGF10 5p13-p12 602115 Lacrimoauriculodentodigital syndrome ITSN2 604464 164860

TABLE-US-00002 TABLE 2 PGX Genes Gene Name ABCG2 ADRA2A ANKK1 APOE COMT CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 CYP3A5 DPYD F2 F5 GRIK4 HTR2A HLA-B MTHFR OPRM1 SLCO1B1 TPMT VKORC1 ABCB1 CYP2B6 CYP2C8 DBH DRD1 DRD4 GABRA6 GABRP HLA-A HCP5 HTR2C ITGB3 KIF6 OPRD1 OPRK1 UGT1A1 UGT2B15 UGT2B7

[0031] The primers used to amplify the sequences are listed in the sequence listing.

[0032] Following standard techniques, the amplified sequences, or amplicons, are purified and a library is made with the purified amplicons. The library contains multiple sequences from different areas of the 374 total genes of interest. The library made from the amplicons is then amplified and sequenced.

[0033] The DNA sequence of the library may be determined by any suitable method. For example, the DNA sequence may be determined by Sanger sequencing (chain termination), pH sequencing, pyrosequencing, sequencing-by-hybridization, sequencing-by-ligation, etc. Exemplary sequencing systems include pyrosequencing (454 Life Sciences), Illumina (Solexa) sequencing, sequencing by ligation (SOLiD, Applied Biosystems), long read sequencing (PacBio or Oxford Nanopore Technologies) and Ion Torrent Systems' pH sequencing system.

[0034] After sequencing, the DNA sequence of the library is mapped with one or more reference sequences to identify sequence variants. For example, the base reads are mapped against a reference sequence, which in various embodiments is presumed to be a “normal” non-disease sequence. The human reference genome sequence (e.g., hg19 or hg38) is generally used as the reference sequence. A number of computerized mapping applications are known, and include GSMAPPER, ELAND, MOSAIK, and MAQ. Various other alignment tools are known, and could also be implemented to map the DNA sequence of the amplicon library.

[0035] Based on the sequence alignments and mapping results, plus available information including, for example, information from human mutational databases and other reference databases, sequence variants in the amplified amplicons are identified. Copy number (i.e., deletions or duplications) may be inferred by analyzing the relative amplicon read depths; deletions will have a statistically significant decrease in relative amplicon read depth spanning the deleted region while duplications will have a statistically significant increase in relative amplicon read depth spanning the duplicated region. Furthermore, any sequence variations, including variants associated with CKD or PGX, disease-associated polymorphisms, benign polymorphisms and other known variants of undetermined significance can be determined to be homozygous, heterozygous, or hemizygous. Any variations in the gene analyzed as compared to the normal control gene can be classified as pathogenic, likely pathogenic, uncertain, likely benign or benign, as recommended by the American College of Medical Genetics (ACMG).

EXAMPLES

Example 1

[0036] DNA was extracted from the whole blood of Patient 1 using known procedures. The extracted sample DNA was then PCR amplified. The complete set of two unique primer pool aliquots were used with the extracted sample DNA. In the present example, the 374 target genes were amplified and analyzed.

[0037] Following amplification of the DNA, the amplicons were purified. A library was then made with the purified amplicons. The library was amplified and sequenced. After sequencing, the sequence of the amplicons from the sample were compared to a control reference sequence comprising the human genome (hg38) sequence.

[0038] A heterozygous mutation in the COL4A4 gene was identified in Patient 1. The patient has a very rare heterozygous nonsense mutation (c.2906C>G, p.Ser969Ter) in exon 32 of the COL4A4 gene. This mutation has a minor allele frequency of 0.00006 in the ExAC database. Truncation mutations such as this one are typically damaging to the protein. It is listed as pathogenic 11 times in the LOVD Gene Variant database. It was found in Alport patients with renal failure between the ages of 20 and 30; four were homozygous and one was heterozygous. This mutation is enriched, particularly in patients of British origin.

Example 2

[0039] DNA was extracted from the whole blood of Patient 2 using known procedures. The extracted sample DNA was then PCR amplified. In the present example, the 374 target genes were amplified and analyzed.

[0040] Following amplification, the amplicons were purified. A library was then made with the purified amplicons. The library was amplified and sequenced. After sequencing, the sequence of the amplicons from the sample were compared to a normal, control reference sequence comprising the hg38 sequence.

[0041] A common polymorphism, c.12627T>G, p.Pro4210Pro, was detected in the PKD1 gene from Patient 2. This polymorphism is present in about 20% of the general population. The polymorphism was detected in 48% of the total reads, indicating this donor is heterozygous for this variant. This polymorphism does not correlate with CKD.

[0042] Although the present invention has been discussed in considerable detail with reference to certain preferred embodiments, other embodiments are possible. Therefore, the scope of the appended claim should not be limited to the description of preferred embodiments contained in this disclosure. All references cited herein are incorporated by reference in their entirety.