Genetics Chromosome and gene aberrations Chromosomal abnormalities Structural Numeric Gene mutatios Rare alleles Polymorphisms Gene mutation - types Normal state DNA ATGCAGGTGACCTCAGTG TACGTCCACTGGAGTCAC Mutation „i DNA ATGCAG TGACCTCAGTG TACGTCG ACTGGAGTCAC AUGCAGGUGACCUCAGU G PROTEIN Met-Gln-Val-Thr-Ser-Val AUGCAG UGACCUCAGUG PROTEIN Met-Gln-Leu-Thr-Ser-Val Examples-hemoglobin Sin sickle heterozygote advantage Gene mutation - types ■ Normal state ■ Mutation „nonsense" ■ DNA ■ DNA ■ ATGCAGGTGACCTCAGTG ■ ATGCAGGTGACCTGAGTG ■ TACGTCCACTGGAGTCAC ■ TACGTCCACTGGACTCAC ■ RNA ■ RNA ■ AUGCAGGUGACCUCAGU ■ AUGCAGGUGACCUGAGUG G ■ PROTEIN . PROTEIN ■ Met-Gln-Val-Thr-Ser-Val . Met-Gln-Val-Thr-Stop _■ Examples: ß° thalasemia Gene mutations- types ■ Normal state ■ Mutation type of trinucleotide expanse ■ DNA ■ DNA ATGCAGGTGACCTCAGTG > ATG(CAGCAGCAG] CAGGTGACCTCAGTG ■ TACGTCCACTGGAGTCAC ■ TAC(GT< >TC] jo GTCC ACTGGAGTCAC ■ RNA ■ RNA ■ AUGCAGGUGACCUGAGUG ■ AUG(CA \G]2„CAGGUGACCUCAGUG ■ PROTEIN ■ PROTEIN ■ Met-Gln-Val-Thr-Ser-Val ■ Met ln]2„Gln-Val-Thr-Ser-Val ■ Examples: Huntington's disease Gene mutations- types Gene mutations- types ■ Normal state a ■ DNA ■ ATGCAGGTGACCTCAGTG ■ ■ TACGTCCACTGGAGTCAC " ■ RNA ■ AUGCAGGUGACCUCAGU G ■ ■ PROTEIN ■ Met-Gln-Val-Thr-Ser-Val Mutation, type „frameshift" DNA ATGCAGGTGAACCTCAGTG TACGTCCAC TGGAGTCAC RNA AUGCAGGUGAACCUCAGUG PROTEIN Met-Gln-Val-Asn-Leu-Ser Examples: Duchenn's muscular dystrophy, p° thalasemia,Tay-Sachs's disease ■ Normal state ■ DNA ■ ATGCAGGTGACCTCA GTG ■ TACGTCCACTGGAGTC AC ■ RNA ■ AUGCAGGUGACCUCA GUG ■ PROTEIN ■ Met-Gln-Val-Thr-Ser-Val ■ Mutation: type ..insertion" ■ DNA ■ ATGCAGGTG-30C ACCTCAGTG ■ TACGTCCAC-30C TGGAGTCAC ■ RNA ■ AUGCAGGUG-30C ACCUCAGUG ■ PROTEIN ■ Met-Gln-Val----------------? ■ Examples: Hemophilia A Gene mutations- types Four basic types of heredity Normal state DNA ATGCAGGTGACCTCAGTG TACGTCCACTGGAGTCAC RNA AUGCAGGUGACCUCAGUG PROTEIN Met-Gln-Val-Thr-Ser-Val Mutation: type ..deletion" DNA ATGCAGGTG TACGTCCAC RNA AUGCAGGUG PROTEIN Met-Gln-Val Examples: small-cystic fibrosis large: Duchenn's muscular dystrophy Monogenic disorders Monogenic disorders ■ Determined by one locus allele. ■ Variant allele which had arosen sometimes in the history replaces original („wild") allele on one (heterozygote) or both (homozygote) chromosomes. ■ Monogenic disorders have a characteristic transfer of the genotypes in families. ■ Rare alleles are associated with monogenic disorders as a „big" factor. ■ Clinical manifestations are observed usually in childhood. ■ Less than 10% are manifested after puberty and only 1% after reproductive age. ■ Prevalence about 0.36%; in 6-8% hospitalizod children some monogenic disorder is suspected. Complex (multifactorial, multigene) diseases ■ They may be interactions of certain gene variants and certain environmental factors (and their combinations) which could be responsible for predisposition for many ■ biological processes ■ evol ■ O 2S. > Genome and environmental factors can be changed by dii > Genome is more im Genome stability vs. genome variability endangered during life time by many repeated DNA replications is preserved by different mechanisms. y seems to be a source of surviving potenciál in changing environmental context. Genome stability ■ Since our genomes are constantly exposed to e: id (e.g. UV radiation) and id (e.g. metabolically generated reactive oxygen species) D dar an impaired ability to detect and/or respond appropriately to these efects can impact on the maintenance of genetic stability. O'DrlscollM: CurrGenomics. 2008 May; 9(3): 137-146 Genome stability ■ There are many examples of human Mendelian disorders defective in the repair of or response to DNA damage. ■ The importance of these pathways is demonstrated by the increase in in and develoDmental abnormalities associated with these conditions. O'DriscollM: CurrGenomics. 2008 May; 9(3): 137-146 Genome instability-copy number variants (CNVs) Recent studies have revealed that DNA segments in sizes from kilobases to megabases can vary in copy number among individuals in a population. These changes in copy number are the result of duplications, deletions, insertions, inversions and complex combinations of rearrangements, and are termed collectively copy number variants (CNVs). van Attikum H, Gasser SM. Trends Cell Biol. 2009 May;19(5):20?-17. Copy number variants The changes in gene copy number are associated with different phenotypes in humans. Perhaps, the most well known example of this is the tn Down syndrome. An increased expression of the genes on chromosome 21 results directly or indirectly in a clinically heterogeneous disorder incorporating impairment, facial dysmorphology, growth retardation, cancer predisposition, microcephaly, heart and skeletal abnormalities Genomic disorders Genomic disorders represent a clinically diverse group of conditions caused by g, orientation of a genomic region containing dosage-sensitive genes. Determining how the o (CNV) affects human variation and contributes to the aetiology and progression of various genomic disorders represents in questions for the future. O'Driscoll M: Curr Genomics. 2008 May; 9(3): 137-146 O'Driscoll M: Curr Genomics. 2008 May; 9(3): 137-146 Gene mutations as a cause of variability in genes (prevalence less than 1% in population as a result of selection pressure and/or „recent" mutation). These mutations represent „great genetic factors" causing es-subjects of cli s (prevalence more than 1% in population, smaller genetic factors in interactions with environmental factors conditioning - subjects of pe Gene mutations as a cause of variability in genes ✓ are generating in somatic cells during the lifetime ✓ are cell and/or tissue specific, without transfer to offspring ■ Mutdtions in QGrm cells •/ they become components of genetic predisposition s they are present in all cells of the individual ✓ they are transfered to offspring Candidate gene and its association with disease ✓ The question is simplier in mendelistic diseases in which a change function of a gene can be easier indentified. ■ IWU 11 lall I possibilities for this strategy: * Linkage analysis s needs examination of genealogy s is evaluating common occurrence of genetic marker and disease in related individuals Genetic studies ■ Candidate s Etiopatogenetic (pathophysiological) approach using for candidate gene selection nalysis s Based on analysis of large sequences of genome Association studies are evaluating common occurrence of genetic marker and disease in unrelated individuals Types of association studies □ case-control (healthy-ill) ? (severity of disease, early onset of disease, risk factors for disease including gender □ genotype-phenotype (e.g.biochemical parameters) DNA markers ■ Thus, it is possible to associate alleles of many polymorphisms with clinical manifestation of the disease and/or with some phenotypes of a disease. ■ Therefore, a certain genotype and/or allele of the polymorphism can represent statistically higher flowed risk for the disease fodds ratioV i Odds ratio (OR): Number of patients with risk genotype x number of healthy individuals with different genotype Number of healthy with risk genotype x number of patients with ottier than risk genotype ^ DNA marker does not have to be causative for the disease. Definitely, the most important characteristic of clinically useful DNA marker must be its high statistic asscociation with a disease and/or its phenotype. Association of a disease with candidate gene variants: an example: I/D ACE Fyhrquist F and Saijonmaa 0, Journal of Internal Medicine 264: 224-236, 2008 RPR, renin/prorenin receptor; Mas, mas oncogene, receptor for Ang 1-7; AT2R, angiotensin type 2 receptor AT1R, angiotensin type 1 receptor, IRAP, insulin-regulated aminopeptidase; Ang IV receptor AMPA, aminopeptidase A; AMPM, aminopeptidase M; ACE, angiotensin-con verting enzyme; ACE2, angiotensin-converting enzyme 2; NEP, neutral endopeptidase. Zygote Embryo Adult organism How does a single egg or zygote become a complete organism with many different tissues and differentiated cells? How can this happen, when the zygote undergoes many rounds of mitosis — mitosis is supposed to produce identical daughter cells? How do Organisms Control the Level of Gene Expression? How do Eukaryotes Control the Level of Gene Expression? ■ Cells must only express genes when needed ■ Gene expression (transcription, translation) takes up large amounts of cellular energy and resources ■ Cells live frugal lifestyles — they conserve energy and resources ■ So genes will only be expressed when their products are needed. ■ Cells of more complex organisms turn on and turn off genes based on the functions of the cells — hence cells differentiate ■ Eukaryotes control genes at almost every level: ■ Regulation of Chromatin Structure ■ Regulation at the transcriptional level ■ Regulation at a post-transcnptional level ■ Regulation at a translational level ■ Regulation at a post-translational level Regulation of Chromatin Structure ■ Histone acetylation prevents DNA from winding tightly around histones, allowing easy access to promoter sites (Deacetylation does the opposite) ■ DNA methylation causes DNA to wind tightly around histones, preventing easy access to gene promoters (Demethylation does the opposite) Histones Histone subunits are: ■ 2 units of H2A ■ 2 units of H2B ■ 2 units of H3 ■ 2 units of H4 Histone HI is not in the core, but acts as a clamp and keeps the linker DNA in place Histones are positively charged, so DNA which is negatively charged, wraps around them Bacteria lack histones, although some archea have them Epigenetic Inheritance Modification of chromatin does not change the DNA, only its expression. However, this modification pattern IS inherited (remember genomic imprinting?) Scientists now believe that certain environmental factors may play a part in promoting chromatin modification that causes expression or suppression of certain genes — e.g. one twin gets schizophrenia and another doesn't. Certain cancers may also be caused that way Epigenetic effects is predominantly and has hi levels of acetylated histone tails. Most mammalian transcription factors have GC-rich binding sites and many have CpGs in their DNA recognition elements. Binding by several of these factors is impeded or abolished by methylation of CpG. 11 Regulation at the transcriptional level ■ Enhancers (proximal and distal) ■ Silencers ■ Transcription factors at promoters ■ General transcription factors ■ Specific transcription factors ■ All these play a role m regulating gene expression. Enhancers increase the rate of a gene's expression and silencers decrease it Transcription factors are needed if the gene is to be expressed at all. Regulation at post-transcriptional level RNA processing — alternative splicing allows certain proteins to be made instead of others (all from the same gene) mRNA Degradation — cytoplasmic nucleases degrade mRNAs so polypeptide synthesis stops. More mRNA is made later, if necessary 5' caps and 3' tails can be removed or changed and this will prevent translation Pre-translational Regulation ■ Certain proteins in the cytoplasm can bind to the mRNA's 5' UTR and prevent ribosomes from binding ■ Any change in mRNA shape will prevent ribosome binding ■ Decreased length of poly-A tail will prevent translation Post-translational Regulation ■ Proteins can be ubiquitinzed and degraded in a proteasome Non-protein-coding RNAs and Gene Regulation ■ MicroRNAs or miRNAs are small non-coding RNAs that were ■ Transcribed from DNA ■ Complexed with a number of proteins ■ These miRNAs have several bases that are complementary to some protein-coding mRNAs ■ The miRNA-protein complex can bind to these protein-coding mRNAs and prevent them from being translated ■ Nucleases eventually degrade the dsRNA Cytoplasmic Determinants ■ Certain molecules such as maternal mRNAs, transcription factors and other proteins are localized in specific cytoplasmic regions of the unfertilized egg or zygote ■ These molecules affect cell fate decisions by segregating into different embryonic cells and controlling distinct gene activities in these cells (specialized transcription factors will only turn on certain genes). ■ Cytoplasmic determinants are also found in some post-embryonic cells, where they produce cytoplasmic asymmetry. ■ In dividing cells, this leads to asymmetric cell division in which each of the daughter cells differentiates into a different cell type. Also called localized cytoplasmic determinants or morphogenetic determinants. Pharmaco-genetic 52 Cytoplasmic Determinants and Cell Fate Pharmacogenetics & Pharmacogenomics Pharmacogenetics & Pharmacogenomics ■ Pharmacogenetics: The role of genetics in drug responses. o F. Vogel. 1959 ■ Pharmacogenomics: The science that allows us to predict a response to drugs based on an individuals genetic makeup. o Felix Frueh, Associate Director of Genomics, FDA ■ P cs: study of individual gene-drug interactions, usually one or two genes that have dominant effect on a drug response (SIMPLE relationship) ■ : study of genomic influence on drug response, often using high-throughput data (sequencing, SNP chip, expression, proteomics - COMPLEX interactions) 14 Relation to genes ■ Almost every pathway of drug metabolism, its transport or activation is influenced by genetic variability ■ Clinical variability in the response ■ The risk of side effects ■ Genotype specific dosage ■ Polymorphic targets drug 58 I - \ii\va 57 10 questions in polygenic disorders s How important are genetic influences in the most common forms of multigene diseases? ^ What is the influence of the environment on the onset of the disease? ^ Which are the most promising approaches to the determination of genetic factors leading to the onset of disease? ^ Which genes have already been selected as candidate genes? ^ Which paths contribute to genetic susceptibility for the disease? ^ How many genes are involved in susceptibility to disease? ^ Are the most common forms of polygenic diseases associated with frequent or rare genetic variability in the population? (hypothesis frequent variations / frequent genetic disease vs. heterogeneous model) ^ Why alleles that are associated with the disease were not eliminated from the population? ^ The importance for the disease-environment interaction genes and genes-genes? What are the implications for pharmacogenetics? 60 Genetic polymorphisms in drug metabolizing enzymes Fhntl -CVP1A1I2 Phase II r^'6' NATS CYP2B5 ^ GST-M / - f>'r. a,i,„- J LtGTe V\ CYP?E1 *"cOUT rpivrr From: Evans WE, Relling MV. Pharmacogenomics: Translating functional genomics into rational therapeutics. Science 286:487-491, 1999. 59 15 Pharmacogenetics: A Case Study Pharmacogenetics: A Case Study Pharmacogenetics: A Case Study Clinically relevant genetic polymorphisms in relation to the side effects of drugs Clinically relevant genetic polymorphisms in relation to the effectiveness of drugs T-i. "TV.* i ......... ^^^n^^^^i all suNlJMisd.™ fflsM m ' T .>.'f :.r ;i.^,£ßm£JhrtS; 1?3 usflinxmdls,. saomaj&lias, Fi^TFT jarcmuSlüa' of ths m£ri3xjrI^uEiiirLs-D MA- D3[^rfb.^*'ljI^J!LSdSr31S5- .§53113 DSEpsaad te fosalnaaiEtr wMi sl^KlaifjriLii^, w: "■■'?.!fc" :--v. 123] liKSiTDSLirii^ .s rx'jVvfilvtaHs-: P< .- '.•!.■;•:: i-.i \ -v ;ji sttefefs-r.- bromcavM!"Jsrtfiir c 1 i&isjstJisa- timm tlhs.n Au&H ■—-l ••srJrmüiiTrB ttel msfls-bciis'srs bd£ EH 65 Glucose-6-phosphate dehydrogenase activity Drugs and Chemicals Unequivocally Demonstrated to Precipitate Hemolytic Anemia in Subjects with G6PD Deficiency Acetanilide Nitrofurantoin Primaquine Methylene Blue Sulfacetamide Nalidixic Acid Naphthalene Sulfanilamide Sulfapyridine Sulfamethoxazole 67 Effects >100 million worldwide -C^E-► R-NOH R- NH, MPO PGH Synthase ERYTHROCYTE NADP+ or \f GSSG(?)^ R-NOH ^ H9bFe NADH 2 NAD+ Sr tA NADPH A R.N0 HgbFe^A*6' orGSH(?)^ J GSH Reactive _^ Splenic Semi-mercaptal Oxygen Sequestration 1 sulfanamide R- NH2 Detoxification Hemolytic Anemia INCIDENCE OFG6PD DEFICIENCY IN DIFFERENT ETHNIC POPULATIONS Ethnic Group Incidence(%) Asiatics Chinese 2 Filipinos 13 Indians-Parsees 16 Javanese 13 Micronesians <1 Iranians 8 Greeks 0.7-3 Persia 15 Cytochrome Oxidase P450 Enzymes ■ 57 Different active genes ■ 17 Different families ■ CYP1, CYP2 and CYP3 are primarily involved in drug metabolism. ■ CYP2A6, CYP2B6, CYP2C9 .CYP2C19, CYP2D6, CYP2E1 and CYP3A4 are responsible for metabolizing most clinically important drugs Polymorphic CYP2B6 CYP2C9 Cytochrome P-450s -ul-lr...... :z::z........- iHIPttHAXI ■>=.;-■-. >|jl I-Halts mid* OnnMM is 3(:i of CnrJMM WS I HypogiywHiif ARBl IrbMdrtan ItaanM 14« i.....>l ■ ■• CYP2C1& CYP2D6 selected . bl ma Meta&oiüer L«J,"H' leiden« Frettjn pump H AmrtriptyHne QNl• ■[ifi-■■■::"i imidi diuefum phe-rtdurbilal CJiromonMrifr 10 15-30^ Allan* ■ -: ■- - : -(hlwohtnlraniln* rfA AA AG 6G Caucasians 19% (N=297) 56% 25% Spanish 32% (N=105) 40% 28% Chinese ^80£) CN=104)>Wj-- 18% 2% African ^^^% Americans fcfch-(N=159) ■E^KE 21% 79% Another Anticoagulant Clopidogrel (Plavix) and CYP2C19 Alleles u )*.r>rtiŕ Rnporic Cop.dogFíl. )D0 mi I,o T ] 5 ■ 1 ! . - ■ i 1 UM EU lU N N-M) IN-11) (N-W) (N>1) PM: with two reduced function alleles IM: one reduced function allele EM= no variant alleles; UM. one or two "17 Interaction with drogs metabolized and/or reactingi with CYP2C9 Competition Enzyme inductor Enzyme inhibitor ASA a většina_ rifampicin fluvoxamin (ostatní SSRI slabí) fenobarbital, fenytoin fenobarbital, fenytoin S-warfarin karta mazapin inhihitniy UMft.P.ftA rarinletáry losartan tolbutamid tolbutamid Cimetidin (slabý) sulfonamidy, dapson aTfilnva amimyfcfitika (slabá) diazepam, tenazepam ritonavir fluoxetin, moclobemid desethylamiodaron zidovudin GENETIC POLYMORPHISMS, MATERNAL SMOKING AND LOW BIRTH WEIGHT (LBW) 65% of all infant deaths occur among LBW infants, while LBW infants account for 7.6% of all live births Reduction in birth wgt among smoking women Genotype CYP1A1 AA CYP1A1 Aa/aa Weight Reduction 252 g 520 g GST1 AA/Aa 285 g GST1 aa_642 g Data from: Wang X, et al. JAMA 287:195-2002, 2002. Metabolic rate b) Geneticky polymorflzmus According to the activity of the enzyme may be a population divided into four main groups - poor metabolisers (PM), intermediate metabolizers (IM), efficient metabolizers (EM), and ultra-fast metabolizers (UM). Mostindividuals among the white population - extensive metabolizers (EM) - the drugs are metabolized by the expected rate. 5-10% of individuals are genetically determined poor metabolisers (PM) - the slow degradation of substances metabolised and are at a higher incidence of adverse events. Intermediate metabolizers (IM) are represented in 10-15% and in long term treatment in response — comparable to PM. Ultra-fast metabolizers (UM) — metabolization is intensive; clinically unresponsive to the usual doses of drugs - 5-10%. Ultrarychli metabolizátori Rychlí metabolizátori Intermediární metabolizátori Pomalí metabolizátori Meta holická aktivita eiizvmu 21 Methotrexate in RA ■ Effectiveness of treatment of rheumatoid arthritis (RA) by methotrexate (MTX) 46% - 65% (ACR20) ■ During treatment with MTX side effects may occur. At least one in 72.9% of patients, severe in 30% of patients. gastrointestinal toxicity (nausea, vomiting, diarrhea, 20% - 65% Hepatotoxicity 10% - 43% oral ulceration 37% alopecia to 4% ■ pulmonary toxicity 2.1% - 8% ■ Bone marrow suppression light 12% ■ pancytopenia 0-8% 86 Methotrexate Methotrexate T Folate Methotrexate Folate Active-Iransport process Dihydtofolate ...... Methotrexate reductase dTMP dUMP Ns, N,0-Methylene-FH4 Folic acid not useful in toxicity Folinicacid N5 formyl FH, should be given which is converted to N5,N10-Methylene -FH, and bypasses the inhibited reductase Adenine, guanine, thymidine, methionine, serine ( lini.jl cllci.1i ■ rfc-i gmoa I M.i HI iTM.IST Un .ii..: MTX irtn into ccl Mm aflcrt rnfaiwc 1» MTX I MTU] R CI,"T ai KtlvHy: i. .■ r ■. MHl c.i .,. ,|, , , i I.. rdptaHn* and mJH&^AJl. h™bdn. Mm(N) Mm likxl MTX cOiun |«J). ur aurn iiiu|iMil) i.. R.A dccraic MTX fe»Kil> <«,. Mo cHhI .m M itw. • M11IIK mclh.kiv.l.lrarliJr.*.lil. r,J«i« li.ii ■.i«l.ii-l..i MIX MktMM K< Ranganathan P, McLeod HL. A&R 20 Methotrexate Halt ■ MTX p»lny I1- K n - i^n,- -n I r:.-. .-. an pen frudlKtn I ] II, Ji . II, 11 ■ { .* ARAB •> C-MJO HLIwwH AJCAR. mjv tmm 5-LTTR 2H-»p May huuk TVMS«vy*k VLTR -M>p Mn ikrnw rYMS nRNA Mn illnl MTX tldc-h V ■■ >lhx1 MTX tdnxl f- j-.... --.-f — -- M!,.i- ... i i - >.i . ... lk>nMil UnJinj illuMi ,-< Mji »11.11 MIX rlku H in* MTXPOi CWIT Atkcu MTXW. k» Mi. dim MTX hOMMy n. 80 Mil dnw MIRK KlnHf. May died MTX hnxMi HI. fC Ranganathan P, McLeod HL. A&R 20* MDR1 MDRl (ATP-binding cassette Bl/multidrug resistance 1) is an efflux pump that transports toxic endogenous substances, drugs and xenobiotics out of cells. It is known to affect susceptibility to many hematopoietic malignancies. ABCB1/MDR1 polymorphisms may either change the protein expression or alter its function, suggesting a possible association between ABCB1/MDR1 single nucleotide polymorphisms (SNP) and clinical aspects of T-cell lymphoma. Therefore, association of two polymorphisms in the gene with clinical staging and therapy was evaluated. (A) An example of an experimentally verified miRNA pharmacogenomic set. miR-125 b inhibits vitamin D receptor (VDR) expression. Why are some gliomas resistant to nitrosourea Evidence suggests this may be the result of an epigenetic phenomenon - one that does not involve a change in DNA ^EmB^I^I MGMT - methylguanine-DNA region of MGMT may silence the gene From: Esteller M, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. NEJM 243:1350-1354, 2000. 93 Glioma with unmethylated MGMT MGMf promote! 1 Exonl Inlionl OS. Co Alkyl group removed by MGMT from the ON A base guanine Glioma with methylated MGMT Methyl MGMTpiornotel Gsne inagbvailon Carmustine cross-links DNA strands, and There is no aclive MGMT to repair it 24 FDA Requires Genetic Tests for Certain Therapies List of FDA Required or Recommended Biomarker Tests in Drug Labels Eiuntirkcr Tciil> Drug Eximpk 1 >l-i Pil^ilIltll-l (n-».l million) CTP2C9 ReconiLUciKted Warfarin (_ LLllAilll.il) G6PD deficiency Eecommttid-eil Diipsnm D 0231 l..I-J 'L"> lUlllltll^ ül-c^mtTAttMltil K.t-''iui-..i-< 0.0000 overex]>K5sloii Trwrnnimih 0.0003 TPMT VLiriiLciii JiL-mTimtriUi'il .\.,nk:i'|Tiiv. 0.1 IAS rPMIvarLitits RcL^mtut tided M I- i ■ ■ ■ i. - L'.-HI TPMT Variants llLLi>]iirTicndc-d [ luo^Li.uuiii: 0.0C12 IICTIAI ■—- Rr-nnrn mended IruiiiM'r.iii 0.0002 Urea cycle enzyme deficiency k.cc4.hh mended \alprak acid ■ \h T- -i. .■ 1 2.766 CVP = iyiLnhiuiii* f4W, t.tit-K = human ijiUtiniLiL |jii-»[h Ittvti rttepi pJiosphale drhydiogLiu». HEfli/iitu ■ human rpldctuiaL growth held r. G4PD = gkKUt-4-K-LtLMOI J 1 I'M . 26