IgA nephropathy At a mean follow-up of 5 years 13% of patients will have some recurrence-r elated renal graft dysfunction and 5% will lose the graft as a result of recurrence. C. NEWSTEAD 574 In the only series in which all patients were biopsied recurrence was found in 58%. Younger patients are at greater risk of recurrence, as are those with a shorter duration of diseas e in the native kidneys. At least one group has demonstrated clinically signi \201 cant po orer graft survival in patients presenting with native cresentic glomerulonephritis. Overal l, graft survival at 5 and 10 years appears to be the same as average. There is no establis hed treatment for preventing or treat- ing recurrent IgA nephropathy, but one group has rep orted a trend towards improved 5 and 10 year graft survival in those prescribed ACEI/ARB th erapy. The use of living related donors for recipients with IgA nephropathy has been the su bject of some controversy. A large registry series found an increased rate of IgA neph- rop athy-related graft loss among zero HLA-mismatched recipients of kidneys from a live donor s ource. This offset the advantage normally seen among zero HLA-mismatched recipients. These authors argued that as overall survival was similar there was no reason to avoid live donor -recipi- ent pairs with zero HLA mismatches. Mesangiocapillary glomerulonephritis type I Where the renal disease is thought to be mediat ed by glomerular deposition of immune complexes generated after exposure, for example to na tive DNA (SLE) or hepa- titis antigens, recurrence would be expected after trans- plantatio n as these antigens would be predicted to persist in the circulation post transplant. Publi shed experience suggests that type I MCGN recurs after renal transplantation in half the pa tients and that this frequency may be even greater in recipients of identical living relate d donor grafts. Recurrence in a second transplant after recurrence in the \201 rst graft is extremely common and once diagnosed has an important detrimental impact on graft surviva l. The risk of recurrence may be considerably less in Japanese patients with a rate of only 3% in one published series. It is not possible to give any evidence-based advice with rega rd to treatment. Antiviral therapy would be logical in patients with persistent hepatitis B antigenemia. Mesangiocapillary glomerulonephritis type II The most comprehensive recent analysis is from the North American Pediatiric database. Recurrent disease was seen in 67% and 5 year graft survival was 50%, compared to 74% on average. There was no correlation between severity of pre- transplantation presentation, pre- or post-transplantation C3 levels and prognosis. Membranous nephropathy In adults the rate of recurrence is 30%. No speci \201 c risk fact or for recurrence has been clearly identi \201 ed. The outcome for graft survival is poor with a 52% graft loss at 10 years. The clinical syndrome seems to be similar to the disease in native kidneys with progressively more severe nephrotic syndrome. The management is usu ally based on extrapolation of data from management of native kidney disease and, similar t o that situation, spontaneous remission, failures of immuno- suppressive treatment as well as clinical response coinci- dent with change of treatment have all been reported. A recent case report of successful use of rituximab requires more experience and longer follow-up. No treatments are known to be effective: there are case reports describing successful use o f plasma exchange and immunosuppression. Systemic lupus erythematosus The rate of recurrent lupus nephritis is 1% at 5 years. Pre-t ransplantation serological parameters are unreliable predictors of the likelihood of recurr ence and also may not accurately re \202 ect disease activity in the post-transplanta- tio n interval. As would be expected given the small number of cases, there are no formal studi es of the management of recur- rent lupus nephritis. Most clinicians would opt for manageme nt that mirrored that of native disease. Anticoagulation during the early perioperative and early post-transplantation phase should be considered for patients with a history of throm bosis or those with the presence of lupus anticoagulant. De novo membranous nephropathy De novo membranous nephropathy is second only to trans- plan t glomerulopathy as a cause of nephrotic syndrome after renal transplantation. The mean tim e of diagnosis is at 5 years post transplanta- tion. The addition of pulsed steroid therap y to the baseline immunosuppression in heavily nephrotic patients makes no obvious impact o n outcome. Systemic sclerosis The evidence base in this condition is extremely small. Most clinicians would manage patients with systemic sclerosis in a similar way to patients with SLE and rec om- mend renal transplantation as a treatment option. The majority would use ACEIs for anti hypertensive control from the outset in patients who had ESRD secondary to scleroderma rena l crisis. Mesangiocapillary glomerulonephritis (MCGN) There are marked histological similarities betw een trans- plant glomerulopathy and MCGN, but immuno \202 uores- cence and electron micros copy can usually discriminate between the two diagnoses. A recent report analyzing the expe rience of a single coun- try (Ireland) concluded that younger age at initial diagnosis and the severity of the native glomerular histology inde- pendently predicted the risk of recur rence, and not the classi \201 cation of MCGN type, with the poorer reputation of type II disease explained by the more aggressive glomeru- lar changes seen in that condition. Further reading Bresin E, et al. Outcome of renal transplantation in patients with non-Shig a toxin-associated haemolytic uremic syndrome: prog- nostic signi \201 cance of genetic ba ckground. Clin J Am Soc Nephrol 2006; 1: 88 \200\22399. Couser W. Recurrent glomerulonephritis in the renal allograft: an update of selected areas. Exp Clin Transplant 2005;3:283 \200\223288. CHAPTER 14.7 Recurrent and de novo disease post renal transplantation 575 American Society of Transplantation: Floege J. Recurrent IgA nephropathy after renal transplantation. Semin Neprol 2004; 24: 287 \200\223291. http://www.a-s-t.org/ American Society of Transplant Surgeons: Gera M, et al. Recurrence of ANCA-associated vasculitis following renal transplantation in the modern era of immunosuppression. Kidney Int 2007; 71: 1296 \200\2231301. http://www.asts.org/ European Society of Pediatric Nephrology (ESPN): Little MA, Dupont P, Campbell E, Dorman A, Walshe JJ. Severity of primary MPGN, rather than MPGN type, determines renal survival and post-transplantation recurrence risk. Kidney Int 2006; 69: 504 \200\223511. http://.espn.cardiff.ac.uk North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS): McDonald SP, Russ GR. Recurrence of IgA nephropathy among renal allograft recipients from l iving donors is greater among those with zero HLA mismatches. Transplantation 2006; 82: 759 \200\223762. https://web.emmes.com/study/ped/ See also Outcome of renal transplantation, p. 576 Early management of transplant recipients , p. 556 Long-term medical complications of renal transplantation, p. 566 Weber S. Tonshoff B. Recurrence of focal-segmental glomeruloscle- rosis in children after r enal transplantation: clinical and genetic aspects. Transplantation 2005; 80: S128 \200\223 S134. Internet resources British Transplantation Society: http://www.bts.org P. TOPHAM 576 Outcome of renal transplantation Renal transplant outcome can be measured in a number of different ways including: \200 \200 \200 \200 \200 \200 Over the past 20 years there has been a 5% and 8% improvement in 5 and 10 year patient surv ival following a deceased donor transplant, but a negligible improvement following a living donor transplant. The principal causes of death beyond 1 year post trans- plant are: \200 \200 \200 graft survival; patient survival; graft function (serum creatinine); number of acute reject ion episodes; days of hospitalization; quality of life. cardiovascular disease; infection; malignancy. Data about transplant outcome usually come from the large registries (for example, the US R enal Database System (USRDS), the Collaborative Transplant Study, and the Australia and New Zealand Dialysis and Transplant registry (ANZDATA)). Patient and graft survival after rena l transplantation have improved signi \201 cantly over the last 25 years although the rate of improvement is now slowing. Graft outcome is traditionally examined in two phases: \200 \200 These three diagnoses account for 67% of deaths in the 45 \200\22364 year age group. Survival bene \201 t of renal transplantation Numerous studies have demonstrated that tran splanted patients have a better long-term outcome than those who remain on dialysis. These studies have often been \202 awed by the inappropriate selection of the comparator group since nontransplanted patients may have more signi \201 cant comorbidity that may prevent them from being transplanted in the \201 rst place. A more appropriate comparison would b e between trans- planted patients and those who are on the transplant wait- ing list but re main untransplanted. The best study of such a comparison indicated that before 106 days pos t transplant the relative risk of death was higher for transplanted patients, mainly becaus e of the risks of the transplantation process (relative risk of death was 2.8 in the \201 rst 2 weeks post transplant). Thereafter transplantation conferred a survival bene \201 t but the overall likelihood of survival did not become equal until day 244. By 3 \200\2234 years post transplant, the relative risk of death was 68% lower in the transplanted group c ompared to those who remained on the transplant list (Fig. 14.8.2). Improved survival was s een in all groups but was particularly high in patients with diabetes mellitus. early (the \201 rst 12 months post transplant); late (any time after the \201 rst 12 mo nths). Short-term outcomes of renal transplantation Graft outcome Currently the 1 year survival ra te for deceased donor allografts is 90%, and 96% for living donor allografts. The 1 year su rvival has steadily improved over the last 20 years from 74% and 89% for deceased donor and living donor allografts respectively in 1987. Early graft loss is usually due to: \200 \200 \200 \200 acute rejection; renal artery thrombosis; patient death; primary nonfunction. Patient outcome The 1 year patient survival rate following a deceased or living donor allog raft is 95% and 98% respectively. The principal causes of death in the \201 rst 12 months are infection and cardiovascular events. Factors affecting renal allograft survival Factors involving both donor and recipient Delay ed graft function Delayed graft function is de \201 ned as the failure to function immedia tely post transplantation with the need for one or more dialysis sessions within a speci \201 ed period (usually one week). Long-term outcomes of renal transplantation Graft outcome Currently the 5 and 10 year graft survival for deceased donor allografts is 68 and 42% respectively. For living donor allogr afts 5 and 10 year graft survival is 80 and 55% respec- tively. The longer-term outcome has also improved over the last 20 years (Fig. 14.8.1). After the \201 rst year post transpl ant the major causes of graft loss are: \200 \200 Deceased donor Living donor 100 80 ) % ( l a v v r u s 60 i patient death; chronic allograft nephropathy. 40 t f a r G 20 Less commonly late acute rejection and recurrent disease can develop. 0 Patient outcome The 5 and 10 year patient survival is 80 and 61% respec- tively for recipie nts of deceased donor allografts, and 89 and 75% for recipients of living donor kidneys. 1987 1991 1994 1997 2000 Fig. 14.8.1 Five-year renal survival probabilities (all transplants that survive beyond 1 y ear) adjusted by age, gender, race and primary diagnosis. (Data from USRDS 2007 Annual Data Report: www.usrds.org/2007/ref/F_tx_outcomes_07.pdf.) CHAPTER 14.8 Outcome of renal transplantation 577 Timing of transplantation Pre-emptive transplantation provides a survival bene \201 t for both allografts and recipients. The relative risk of graft loss and patient death have been shown to be reduced by 27% and 31% respectively follow- ing pre-emptive living donor trans plantation. Similar bene- \201 t has been described in deceased donor transplantation. Th e length of time on dialysis before transplantation also impacts on outcome. For example it has been demon- strated that 10 year allograft survival in living donor recipi- ents trans planted pre-emptively was 75%. 10 year graft survival for those dialysed for: \200 \200 \200 \200 Delayed graft function is associated with: \200 \200 \200 \200 worse graft survival; worse graft function; higher risk of recipient death; higher rate of acute rejection. The increased risk of acute rejection may be because ischemia-reperfusion injury enhances t he immunogenicity of the graft. The rates of delayed graft function remain 20% for decease d donor allografts and 3% for living donor allografts. The risk of delayed graft function i ncreases with: \200 \200 \200 \200 increasing donor age; longer cold ischemia time; Black recipients; presence of >50% panel r eactive antibodies. 0 \200\2236 months was 62%; 6 \200\22312 months was 56%; 12 \200\22324 months was 54%; >24 months was 49%. HLA matching For deceased donor allografts better HLA matching is associated with improved survival. For six antigen-matched kidneys this bene \201 t outweighs the detrimental effec t of prolonged cold ischemia. The fore- most organ-sharing systems will sanction the transp ortation of fully matched organs over long distances. For mismatched grafts however, there is a progressive fall in graft survival with increasing cold ischemia time (1 \200\2232% pe r 12 h increase in cold ischemia time). There is a progressive fall in graft survival with increasing mismatch. For living donor allografts the effect of HLA matching is much less pr onounced (although there is still a bene \201 t seen in two haplotype-matched grafts). Thi s is possibly due to the absence of signi \201 cant ischemic injury. The same effect was observed in recipients of deceased donor kidneys. The bene \201 cial e ffect of pre-emptive transplantation may be due to the: \200 avoidance of the adverse consequences of dialysis (predominantly the development of cardiov ascular dis- ease and malnutrition); reduced incidence of delayed graft function; reduced i ncidence of acute rejection in the \201 rst 6 months post-transplantation; effect of soci oeconomic factors. Pre-emptive transplan- tation tends to be undertaken in younger, Caucasi an, and educated patients with fewer HLA mismatches. \200 Donor factors Donor age Increasing age of the donor reduces long-term allograft survival. T he optimal donor age is 18 \200\22334 years. The 5 year survival of an allograft from a don or aged 18 \200\22334 years is 78% compared to 55% for a kidney from a donor aged >65 years . This is an increasingly important issue because all registries have reported that the ave rage age of donors is increasing, presumably due to the general shortage of kidneys for don ation and the acceptance of more marginal kidneys to increase the size of the donor pool. Cytomegalovirus status of the donor and recipient There is a small but demonstrable effect of CMV status on allograft outcome. CMV \200\223 donor/CMV \200\223 recipient combina- tion s have better outcomes than CMV+ donor/CMV \200\223 recipient combinations. This difference is likely mediated by overt CMV infection/ disease, but subtle effects of CMV on the immun e system may be involved. CMV prophylaxis has been shown to substantially reduce the incide nce of acute rejection. Type of kidney Living donor vs deceased donor. Living donor grafts are supe- rior to deceas ed donor grafts. This is likely to be because of: \200 \200 \200 \200 \200 the absence of signi \201 cant cold ischemia; the absence of brain death; the better gener al health of the donor; higher nephron mass; possibly the shorter wait before transplantati on. 4.0 h t a e d 2.84 f o k s i r Recipient concordance with immunosuppressive medica- tion may also be better in the living donor setting. Heart-beating vs nonheart-beating deceased donors. Allografts from a nonhear t-beating deceased donor source do less well than those from a heart-beating donor. The com parative 1 year graft survival is 72% and 84% for nonheart-beating and heart-beating source s respectively. Longer-term survival appears to be very similar to that seen with heart-bea ting donors. 1.0 e v i t a l e R 0.32 0.25 0 106 183 244 365 548 Days post transplantation Fig. 14.8.2 Adjusted relative risk of death among 23 275 recipients of a \201 rst cadaver ic transplant. (Reproduced with permission from Wolfe et al., 1999.) P. TOPHAM 578 \200 Extended criteria donors. Due to the mismatch between the supply of and demand for kidneys for transplantation, extended criteria donor kidneys are being used increas- ingly to expan d the donor pool. The extended criteria include kidneys from: \200 enhanced immune responsiveness; poorer HLA-matching (with a predominantly Caucasian donor p ool); a higher prevalence of hypertension; altered immunosuppressive pharmacokinetics and p harmacodynamics; socioeconomic factors may also play a role. \200 donors aged >60 years; or \200 By contrast, Asian and Hispanic recipients in the USA have better graft outcomes than Cauca sians. donors aged >50 years with two of the following fea- tures: a history of hypertension; deat h by CVA; terminal creatinine levels >133 mol/L. Recipient gender There are con \202 icting data about the in \202 uence of gender on rena l allograft outcomes, with studies demonstrating both better outcomes in females who receiv e deceased donor kidneys, and in males who receive living donor kidneys. Of relevance to tr ansplantation, females are often sensi- tized to HLA and non-HLA antigens through pregnancy . The use of extended criteria donor kidneys is associated with worse graft outcome with graf t survival inversely cor- relating with the quality of the kidney (although the quality of a kidney can be hard to determine in practice). It is estimated that the relative risk of g raft failure with extended criteria grafts compared to those from an \200\230ideal \200 \231 source is 1.7. Acute rejection In both living and deceased donor transplants, a single episode of acute re jection reduces the 5 year graft survival by 10%. The effect of acute rejection is cumulat ive: The 10 year graft survival following more than one episode of acute rejection was 54% compared to 88% and 96% following one or no episode of acute rejection respectively. Donor gender There is some evidence that grafts from deceased female donors have shorter su rvival, particularly when trans- planted into male recipients. This is likely due to the re la- tively smaller size of the kidneys and nephron underdosing (see below). Ischemia reperf usion injury Longer cold ischemia increases the risk of delayed graft function and reduces long-term graft survival. This risk is particularly high when cold ischemia exceeds 18 h an d when kidneys are sourced from donors aged >60 years. There is some evidence that pulsatil e perfusion of har- vested kidneys may improve long-term survival compared to simple cold s torage (10% graft survival bene \201 t at 2 years post transplantation). HLA antibodies Anti-HLA antibodies are present in 20% of renal transplant recipients. The p resence of anti-HLA antibodies doubles the risk of graft failure by 1 year, and the de novo formation of anti- HLA antibodies trebles this risk. Highly sensitized patients are less l ikely to be transplanted because of a positive pretransplant crossmatch. Nonetheless the pr esence of panel-reactive antibodies (PRA) increases the risk of acute rejection and graft l oss. Thus the 5 year deceased donor allograft survival rates were shown to be: \200 \200 \200 Nephron mass The transplantation of a kidney that contains an inadequate number of nephrons (nephron underdosing), may reduce long-term allograft survival. This may occur when kidney s from old or very young donors are used, or when a normal kidney is transplanted into a la rge (>100 kg) recipient. The worse outcome of female-to-male transplantation may also be pa rtially explained on this basis. 70% for 0 \200\22310% PRA; 65% for 10 \200\22379% PRA; 63% for >80% PRA. There is evidence from a variety of sources that non-HLA immunity is important in determini ng the outcome of patients with high level PRA. Recipient factors Recipient age Graft survival is less good at the extremes of age. In youn g patients grafts are more likely to be lost due to technical issues such as vessel thrombo sis, and acute rejection. In older patients (>65 years) the commonest cause of graft loss i s death with a functioning kidney. There is also evidence that acute rejection is less comm on in elderly patients and therefore less aggressive immuno- suppression may be appropriate in such patients. Body mass index Obesity is associated with: \200 \200 \200 \200 more transplantation-related surgical complications; more delayed graft function; higher mo rtality; poorer graft survival. It has been suggested that, where possible, obese potential recipients should lose weight t o achieve a BMI <30 kg/m2 prior to transplantation. Nonetheless, transplanted obese patient s derive a survival bene \201 t when compared to those who remain on dialysis. There is al so evidence for increased mortality and graft loss in those with a low BMI (<18 kg/m2). Rec ipient hypertension Hypertension adversely affects long-term allograft and patient survival . Recipient race In the USA, Black recipients have poorer deceased donor allograft survival t han Caucasian recipients. Potential explanations for this observation include: \200 \200 a higher incidence of delayed graft function; a higher incidence of acute rejection; CHAPTER 14.8 Outcome of renal transplantation 579 \200 \200 \200 \200 \200 use of younger, generally healthier donors; zero HLA mismatching; improved organ preservati on; reduced cold ischemic time; nephron dosing; matching of donor/recipient with regard to nephron \200\230dose \200\231; minimized used of calcineurin inhibitors; RAS blockade; imp roved general medical care (including aggressive lipid-lowering and blood pressure control) . It has been estimated that for every 10 mmHg rise in BP the relative risk of allograft fail ure is 1.3. Recipient dyslipidemia Some studies have suggested that hyperlipidemia and/or hypertriglyce ridemia are associated with poorer allograft outcomes. There are preliminary data indicatin g that lipid-lowering treatment with HMG-CoA reductase inhibitors may improve allograft out come. \200 \200 Recurrent or de novo glomerular disease In patients with ESRD due to glomerulonephritis the 10 year incidence of graft loss due to recurrent glomeru- lonephritis is 8.4%. It is the t hird most common cause of graft failure behind chronic rejection and death. It is likely th at as overall allograft survival improves, graft loss due to recurrent or de novo glomerula r disease will become a more signi \201 cant problem. Further reading Meier-Kriesche HU, Schold JD, Kaplan B. Long-term renal allograft survival: have we made signi \201 cant progress or is it time to rethink our analytic and therapeut ic strategies? Am J Transplant 2004; 4: 1662 \200\2231668. Pascual M, Theruvath T, Kawai T, Tolkoff-Rubin N, Cosimi AB. Strategies to improve long-ter m outcomes after renal transplan- tation. N Engl J Med 2002; 346: 580 \200\223590. Immune response gene polymorphisms Genetic variations in the ability of the immune system t o mount an immune response to an allograft may affect renal transplant outcome. Candidate p olymorphic genes would include those encoding cytokines, chemokines, adhesion molecules and their respective receptors. It has been suggested for instance that recipients who harbor homozygous inactivating mutations of the CCR5 gene have a 20 year graft survival of 90% com pared to 25% for other allografts. These studies are inconclusive since they are often retr o- spective and do not account for other factors that may in \202 uence graft outcome. Tang IY, Meier-Kriesche HU, Kaplan B. Immunosuppressive strate- gies to improve outcomes of kidney transplantation. Semin Nephrol 2007; 27: 377 \200\223392. Wolfe RA, Ashby VB, Milford EL, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplanta- tion, and recipients of a \201 rst cadaveric transplant. N Engl J Med 1999; 341: 1725 \200\2231730. Internet resources United States Renal Data System (USRDS): www.usrds.org/ CTS \200\223 Collaborative Transplant Study: www.ctstransplant.org/ Australia and New Zealand Dialysis and Transplant Registry: Proteinuria The degree of proteinuria correlates with renal allograft outcome. Proteinuria is undoubtedly a re \202 ection of the severity of the underlying renal injury (chronic al lograft nephropathy or recurrent/de novo glomerular disease), but there is also evidence th at proteinuria per se can promote further renal injury. There is a large body of evidence t hat RAS blockade in native kidneys slows renal disease progression although such data are l acking in the renal transplant setting. Nonetheless RAS blockade with ACEI and/or ARB shoul d be used in proteinuric renal allograft recipients. Renal function should be monitored clo sely after introduc- ing such agents since catastrophic renal failure can occur when there is undiagnosed transplant renal artery stenosis. www.anzdata.org.au/ UK Renal Registry: www.renalreg.com/ See also Transplant surgery and surgical complications, p. 552 Early management of transpla nt recipients, p. 556 Immunosuppression for renal transplantation, p. 560 Long-term medical complications of renal transplantation, p. 566 Recurrent disease and de novo disease post renal trans- plantation, p. 572 Strategies to improve renal transplant outcome Measures that are likely to improve renal transplant out- come include: \200 \200 increased use of living donor kidney allografts; pre-emptive transplantation; This page intentionally left blank 581 Chapter 15 Inherited renal disease Chapter contents Investigation of inherited renal disease 582 Polycystic kidney disease in children 586 Auto somal dominant polycystic kidney disease 590 Tuberous sclerosis 598 von Hippel \200\223Lind au disease 600 Nephronophthisis 602 Thin membrane nephropathy 606 Alport \200\231s syndrome 608 Nail \200\223patella syndrome 614 Congenital and infantile nephrotic syndrome 616 Fabr y disease 620 Other metabolic storage disorders with renal involvement 624 Cystinosis 628 P rimary hyperoxalurias 632 Inherited disorders of purine metabolism and transportation 636 R are syndromes with renal involvement 640 F. HILDEBRANDT AND M. T. F. WOLF 582 Investigation of inherited renal disease Gene \200\223protein \200\223phenotype The \202 ow of genetic information In every cell o f the human body the nucleus contains 35 000 structural genes that represent the entirety of genetic information. Tissue speci \201 city, external stimuli, developmental stage and the current functional state of a cell determine which of these genes are actively expresse d, i.e. a gene is transcribed into messenger RNA (mRNA), which is translated into a protein , which through its func- tion, determines a phenotype of a cell (Fig. 15.1.1). been identi \201 ed that are responsible for renal tumors, renal tubular disorders and nep hrolithiasis. Molecular genetic diagnostics In a monogenic disease the speci \201 c gene defect represen ts the primary cause of this disorder. Gene identi \201 cation allows for unequivocal mole cular genetic diagnosis. In molecular genetic diagnostics two approaches are discerned: Dir ect genetic testing: which can be applied if the disease- causing gene is known. Indirect g enetic testing: which is employed if the causative gene is unknown but has been localized t o a specific chromosomal region by linkage analysis. Monogenic diseases Monogenic diseases are caused in any one patient by muta- tions in one g ene only. In recessive diseases a mutation is on both parental chromosomes, whereas in domi nant dis- orders a defect on one parental chromosome is patho- genic. Several hundred renal diseases are \200\230monogenic diseases \200\231. Monogenic diseases are usually rare dis orders in the range of 1:1 000 to 1:1 000 000 individuals. Direct genetic testing Direct sequencing In diseases in which the responsible gene is known , rele- vant mutations may be detected directly in affected indi- viduals following ampli \201 cation of exons by polymerase chain reaction and consecutive direct sequencing. Condit ions that need to be met to allow for direct molecu- lar genetic diagnostics are: 1. The di sease gene must have been identi \201 ed. 2. The functional relevance of mutations detecte d must be proven by absence from 100 healthy controls, or by a functional test. Polygenic diseases In polygenic diseases, for a given patient, only the actions of differen t gene defects together lead to the disease pheno- type through interaction of the encoded proteins. Polygenic disorders are usually frequent diseases occurring in the range of up to 1:5 (e.g. essential hypertension) to 1:1000 individuals. 3. The mutation may have been shown to be responsible Genes Positional cloning of disease genes Family studies and haplotype analysis The set of 46 human chromosomes consists of two sex chromosomes and a double set of 22 autosomes, one set of which is inherited from the father and one from the mother. Crossovers between paren tal homologous chro- mosomes can occur in meioses of the gametes that lead to a newborn chi ld. In family studies these crossovers can be observed by examination of genomic DNA from a ffected individuals. This method is called haplotype analysis (Fig. 15.1.2). This critical region is narrowed down by iden- tifying more crossovers in additional families, until only a few genes can be accommodated in this region. The exons of these genes will have to be e xamined by direct sequenc- ing of DNA in affected individuals. Identi \201 cation of muta- tions in a speci \201 c gene will then identify this gene as responsible for the disease. for the disease phenotype in the literature. 4. If there are multiple affected individuals, the mutation must segregate within the pedigree. For molecular genetic testing prior genetic counseling is mandatory, and ethical guidelines should be observed. The GeneTests database provides help in \201 nding laborato- ries al l over the world that offer molecular genetic diag- nostics for inherited renal diseases. Indirect genetic testing If the causative gene for a monogenic disorder is unknown, but a g ene locus has been localized to a chromosomal region by positional cloning, indirect geneti c testing by hap- lotype analysis at this gene locus can be performed for diagnostics (see also Fig. 15.1.2). Electronic databases Valuable resources are available from electronic databases on the Inte rnet regarding genomic, proteomic and disease- related information. www.ncbi.nlm.nih.gov is a useful place to start. Single nucleotide polymorphisms There are 3 \200\2235 million nucleotide positions in the human genome at which individuals may differ (single nucleotide polymorphisms SNPs) from ea ch other ( 0.1% of the genome). These SNPs can be detected by DNA microarray analysis. They are used for linkage analysis and are replacing microsatellite markers. This approach has already been very helpful for studies into polygenic diseases. Animal models of genetic renal disease Transgenic mouse models are very helpful for the stu dies of human genetic disorders, and are greatly facilitated by the mouse genome sequence. Mouse models of renal dis- ease have become available through identi \201 cation of dis- e ase phenotypes that are based on spontaneously arisen mutations. In addition, mouse models of human renal genetic disease have been generated by targeted disrup- tion of speci \201 c disease-causing genes. Known monogenic causes of renal disease In the past few years the genes responsible for man y monogenic renal disorders have been identi \201 ed by posi- tional cloning. There are re nal cystic diseases, such as autosomal dominant polycystic kidney disease (ADPKD), autosoma l recessive polycystic kidney disease (ARPKD), and nephronophthisis. There are monogenic gl omerular dis- eases, e.g. mutations in the podocin gene in steroid-resistant nephrotic synd rome, and mutations in speci \201 c genes have Zebra \201 sh As a lower vertebrate the zebra \201 sh (Danio rerio) has been a very usefu l model for the study of kidney organogenesis and human renal cystic diseases. CHAPTER 15.1 Investigation of inherited renal disease 583 antibody has been coupled to a color detection tool, e.g. by using horseradish peroxidase o r enhanced lumines- cence, which allows detection of the secondary antibody in its bound st ate to the primary antibody, and thereby detec- tion of the band representing the protein o f interest. If a suitable antibody is available, Western blotting thus allows detection of a protein of interest out of a complex protein mixture, such as total cell lysates from cel l culture. As an example, Western blotting can be employed for detection of proteins from c o-immunoprecipitation experiments (Fig 15.1.3). In very complex protein mixtures, size sepa ration by SDS \200\223PAGE alone may not yield suf \201 cient resolution. Under these circ umstances 2-dimensional gel electrophoresis may be used. In this method proteins are \201 rst separated by their characteristic isoelectric point through isoelectric focusing in a tube gel. Thereafter the linear tube gel is loaded onto an SDS \200\223PAGE gel and pro- te ins are size-separated in a direction perpendicular to the direction for isoelectric focusi ng. Thereby 2-dimensional size separation is achieved according to two independent characte ristics: isoelectric point and molecular weight. Flow of Genetic Information ( \200\235Central Dogma \200\235) Gene mRNA Protein Phenotype AAAA Monogenic Disease Gene defect Disease \200\234Pathogenesis \200\235 AAAA = Etiology Fig. 15.1.1 Central dogma of molecular biology and its relation to monogenic disease mechan isms. Protein \200\223protein interaction Co-immunoprecipitation Protein \200\223protein interact ions can be demonstrated by co-immunoprecipitation experiments. Both proteins or fragments thereof are coexpressed in a suitable cell line through transfection of this cell line with expression- plasmids containing inserts encoding these proteins. If no cognate antibody is available for these proteins, they can be expressed as fusion proteins containing a peptid e tag for which antibodies are available, such as myc-tags or FLAG-tags. After transfection and cell lysis the lysates are incubated with the antibody directed against one protein or its tag, antibodies with bound protein are precipitated and the precipitate isolated. The precipitate is then size- separated by SDS \200\223PAGE, and protein \200\223protein intera ction partners are detected by Western blot analysis of the precipitate (Fig. 15.1.3A). The results usually have to be corroborated in the reciprocal form, i.e. if protein \200\230A \200\231 precipitated with protein \200\230B \200\231, protein \200\230B \200\231 should also co-precipitate with protein \200\230A \200\231 (Fig. 15.1.3B). Fig. 15.1.2 The principle of haplotype analysis. Pull-down assays (in vitro assays) In so-called \200\230pull-down assays \200\231, in vitr o protein interaction is examined by adsorption of one protein-binding part- ner as fusion protein to a solid phase, e.g. in a column of this solid phase material. The column is then equili- brated with a lysate that contains the binding partner under high salt conditions, allowing the binding partner to interact. Other nonbinding proteins from the lysate are wa shed out at high salt. Finally the binding partner is eluted under low salt conditions and detected, e.g. by Western blot analysis. In this way interaction of the proteins poly- cyst in 1 and 2, the genes of which cause autosomal poly- cystic kidney disease types 1 and 2, r espectively, has been demonstrated. Caenorhabditis elegans Many genes which cause renal cystic disease (PKD1, PKD2, BBS, NPHP) show conservation of sequence homology and of function in the nematode Caenorhabditis elega ns (C. elegans). The genome has been fully sequenced and the cell lineage of every cell in development of C. elegans can be followed under a microscope using Nomarski optics. Therefo re, C. elegans is a very useful model for the study for physiologic and patho- physiologic mechanisms. Proteins Generation and use of antibodies as molecular probes Western blotting In Western b lotting proteins are \201 rst size-separated by sodium dodecyl sulfate \200\223polyacryla mide gel electro- phoresis (SDS \200\223PAGE), then electroblotted onto a suitable membrane to yield a spatial representation of the result from size separation. This representation can then be visu- alized by staining with amido black or Ponceau S. The membrane is overlai d with \200\230primary \200\231 antibody directed against the protein of interest. Unbound antibody is then washed away, and secondary antibodies recognizing the Fc fragments of the primary antibody are added. The secondary Mapping of protein \200\223protein interaction domains In vitro protein \200\223protein int eraction assays from in vitro interaction assays or from co-immunoprecipitation can be used to map protein \200\223protein interaction domains. For this purpose speci \201 c constru cts containing the full length of the protein and others that contain only fragments of the pro- tein are tested for interaction. Depending on the result, regions necessary for inter action can be delineated. F. HILDEBRANDT AND M. T. F. WOLF 584 (a) (b) Fig. 15.1.3 Co-immunoprecipitation of nephrocystin-1 and p130CAS. (a) Myc-tagged murine p13 0CAS and FLAG-tagged human nephrocystin were co-expressed in baby hamster kidney cells. Whe n nephrocystin was precipitated (IP) with the anti-FLAG- antibody, p130CAS was shown in a W estern blot to co-precipitate using an anti-myc-ab (right lane; CAS). Also shown is 1/20 of the cell lysate input as positive control (left lane), and IP with an irrelevant antibody (middle lane). Strong bands at 60 kDa are immunoglobulin heavy chains. (b) Vise versa, if p 130CAS was precipitated, nephrocystin-1 (NPHP1) co-precipitated, as detected by Western blo t using an anti- FLAG-tag antibody. Identi \201 cation of new protein \200\223protein interaction partners In order to identif y novel binding partners to a protein or peptide the yeast-2-hybrid system can be employed. In this way the protein p130CAS has been identi \201 ed as a binding partner to nephrocys tin, the product of the NPHP1 gene, which if defective gives rise to juvenile nephronophthi sis type 1 (Fig. 15.1.3).