Molecular genetics of nephronophthisis and medullary cystic kidney disease

Nephronophthisis (NPH) and medullary cystic kidney disease (MCKD) constitute a group of renal cystic diseases that share the macroscopic feature of cyst development at the corticomedullary border of the kidneys. The disease variants also have in common a characteristic renal histologic triad of tubular basement membrane disintegration, tubular atrophy with cyst development, and interstitial cell infiltration with fibrosis. NPH and, in most instances, MCKD lead to chronic renal failure with an onset in the first two decades of life for recessive NPH and onset in adult life for autosomal dominant MCKD. There is extensive genetic heterogeneity with at least three different loci for NPH (NPHP1, NPHP2, and NPHP3) and two different loci for MCKD (MCKD1 and MCKD2). Juvenile nephronophthisis, in addition, can be associated with extrarenal organ involvement. As a first step toward understanding the pathogenesis of this disease group, the gene (NPH1) for juvenile nephronophthisis (NPH1) has been identified by positional cloning. Its gene product, nephrocystin, is a novel protein of unknown function that contains a src-homology 3 domain. It is hypothesized that the pathogenesis of NPH might be related to signaling processes at focal adhesions (the contact points between cells and extracellular matrix) and/or adherens junctions (the contact points between cells). This hypothesis is based on the fact that most src-homology 3-containing proteins are part of focal adhesion signaling complexes, on animal models that exhibit an NPH-like phenotype, and on the recent finding that nephrocystin binds to the protein p130(cas), a major mediator of focal adhesion signaling.     

Nephrocystin specifically localizes to the transition zone of renal and respiratory cilia and photoreceptor connecting cilia

Nephronophthisis (NPHP) is a hereditary cystic kidney disorder that causes renal failure in children and young adults and can be associated with various extrarenal disorders, including retinitis pigmentosa. Six NPHP genes, whose functions are disrupted by autosomal recessive mutations in patients with NPHP, have been identified. The majority of patients with NPHP carry homozygous deletions of NPHP1 encoding nephrocystin. Previous data indicate that nephrocystin forms a complex at cell junctions and focal adhesions. Here, it is shown that nephrocystin specifically localizes at the ciliary base to the transition zone of renal and respiratory cilia and to photoreceptor connecting cilia. During in vitro ciliogenesis of primary human respiratory epithelial cells, nephrocystin can be detected first with a diffuse cytoplasmic localization as soon as cell polarization starts, and translocates to the transition zone when cilia are formed. In columnar respiratory cells, nephrocystin is attached tightly to the axonemal structure of the transition zone at a region that contains the calcium-sensitive cilia autotomy site. In patients with homozygous NPHP1 deletions, nephrocystin is absent from the entire respiratory cell, including the transition zone, which might be of interest for future diagnostic approaches. Cilia formation is not altered in primary nephrocystin-deficient respiratory cells, which is consistent with previous findings obtained for the Caenorhabditis elegans ortholog. In addition, it is shown that the localization pattern of intraflagellar transport proteins and nephrocystin differs, suggesting distinct functional roles. In conclusion, nephrocystin deficiency or dysfunction at the transition zone of renal monocilia and the photoreceptor connecting cilium might explain renal failure and retinal degeneration that are observed in patients with NPHP1.     

Expression and phenotype analysis of the nephrocystin-1 and nephrocystin-4 homologs in Caenorhabditis elegans

Nephronophthisis (NPHP), an autosomal-recessive cystic kidney disease, is the most frequent genetic cause of end-stage renal failure in children. NPHP types 1 and 4 are caused by mutations in NPHP1 and NPHP4, encoding the proteins nephrocystin-1 and nephrocystin-4, respectively. Nephrocystin-1 and nephrocystin-4 are expressed in primary cilia of renal epithelial cells. NPHP1 and NPHP4 are highly conserved in Caenorhabditis elegans. However, this species does not have a kidney but an excretory system that consists of an excretory cell, an excretory gland cell, a duct cell, and a pore cell. Therefore, cell type-specific expression pattern and function of the nephrocystin homologs in C. elegans were of interest. Expression of green fluorescence protein fusion constructs that contain the C. elegans promoter regions for nph-1 and nph-4 was not found in the excretory system but in ciliated sensory neurons of the head (amphid neurons) and the tail in hermaphrodites (phasmid neurons) and males (sensory ray neurons). As the knockout phenotype for the PKD homologs lov-1 and pkd-2 shows impaired male mating behavior, RNAi knockdown animals were analyzed for this phenotype. A similar phenotype was found in the nph-1 and nph-4 RNAi knockdown animals compared with the lov-1 and pkd-2 knockout phenotype. Thus, it is suggested that renal cyst-causing genes may be part of a shared functional module, highly conserved in evolution. The NPHP homologs may be necessary for initial assembly of the cilium, whereas the polycystic kidney disease homologs may function as sensory transducers.     

Nephronophthisis

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease and the most frequent genetic cause of end-stage renal disease up to the third decade of life. It is caused by mutations in 11 different genes, denoted nephrocystins (NPHP1–11, NPHP1L). As an increasing number of these genes are identified, our knowledge of nephronophthisis is changing, thereby improving our understanding of the pathomechanisms in NPHP. Recent publications have described ciliary expression of nephrocystins together with other cystoproteins, such as polycystins 1 and 2 and fibrocystin. These findings have shifted our focus to a pathomechanism involving defects in ciliary function (ciliopathy) and planar cell polarity (PCP). In addition, discoveries of new nephrocystin genes have shown that the disease spectrum of NPHP is much broader than previously anticipated. Different forms of mutations within the same NPHP gene can cause different disease severity. In this review, we highlight the different hypotheses on the pathomechanisms for NPHP and underline the clinical variability of this disease. The clinical spectrum has become even more complex with the possibility of oligogenicity in NPHP.     

Urinary concentration defects and mechanisms underlying nephronophthisis

The cystic kidney disease nephronophthisis (NPHP) is the commonest genetic cause of end-stage renal failure in young people and children. Histologically the disease is characterized by interstitial fibrosis, tubular atrophy with corticomedullary cyst development and disruption of the tubular basement membrane. Affected children present with polydipsia and polyuria, secondary to a urinary concentration defect, before these structural changes develop. Recently, molecular genetic advances have identified several genes mutated in NPHP, providing novel insights into its pathophysiology for the first time in decades. Here we review the normal physiological mechanisms of urinary concentration and explain, in the context of recent discoveries, the possible mechanisms underlying urinary concentration defects in patients with NPHP. The pattern of a ciliary and adherens junction subcellular localization of nephrocystin proteins is discussed. Recent animal models of cystic kidney disease and treatment with vasopressin V2 receptor antagonists are reviewed and a hypothesis regarding urinary concentration defects in NPHP is proposed. Understanding the cellular mechanisms underlying NPHP and other cystic kidney diseases will provide the rationale for therapeutic interventions in this disease. Early urinary concentration defects provide both a clue to clinical diagnosis of NPHP and potential therapeutic targets for pharmacological treatment of this condition.     

Human adolescent nephronophthisis: gene locus synteny with polycystic kidney disease in pcy mice

In a large Venezuelan kindred, a new type of nephronophthisis was recently identified: Adolescent nephronophthisis (NPH3) is a late-onset recessive renal cystic disorder of the nephronophthisis/medullary cystic group of diseases causing end-stage renal disease at a median age of 19 yr. With the use of a homozygosity mapping strategy, the gene (NPHP3) was previously localized to chromosome 3q22 within a critical interval of 2.4 cM. In the current study, the NPHP3 genetic region was cloned and seven genes, eight expressed sequence-tagged sites, and seven microsatellites were physically localized within the critical disease interval. By human-mouse synteny analysis based on expressed genes, synteny between the human NPHP3 locus on chromosome 3q and the pcy locus on mouse chromosome 9 was clearly demonstrated, thus providing the first evidence of synteny between a human and a spontaneous murine renal cystic disease. By fluorescence in situ hybridization the chromosomal assignment of NPHP3 to chromosome 3q21-q22 was refined. Renal pathology in NPH3 was found to consist of tubular basement membranes changes, tubular atrophy and dilation, and sclerosing tubulointerstitial nephropathy. This pathology clearly resembled findings observed in the recessive pcy mouse model of late-onset polycystic kidney disease. In analogy to pcy, renal cyst development at the corticomedullary junction was found to be an early sign of the disease. Through cloning of the NPH3 critical region and mapping of expressed genes, synteny between human NPH3 and murine pcy was established, thus generating the hypothesis that both diseases are caused by recessive mutations of homologous genes.     

New insights: nephronophthisis-medullary cystic kidney disease

Nephronophthisis (NPH) and medullary cystic kidney disease (MCKD) constitute a group of renal cystic diseases, which share a common characteristic renal histologic triad of tubular basement membrane disintegration, tubular atrophy with cyst development, and interstitial cell infiltration with fibrosis. The different disease variants lead to chronic renal failure with onset at characteristic age ranges for recessive NPH and dominant MCKD. There is extensive gene locus heterogeneity with at least three different loci for nephronophthisis (NPHP1, NPHP2, and NPHP3) and two different loci for MCKD (MCKD1 and MCKD2). Juvenile nephronophthisis, in addition, can be associated with extrarenal organ involvement. We have identified by positional cloning the gene (NPHP1) for juvenile nephronophthisis (NPH1), as a first step towards understanding the pathogenesis of this disease group. Its gene product, nephrocystin, is a novel protein, which contains a src-homology 3 (SH3) domain. We put forward a hypothesis that the pathogenesis of NPH might be related to signaling processes at focal adhesions (the contact points between cells and extracellular matrix) and/or adherens junctions (the contact points between cells). Received: 13 July 2000 / Revised: 1 October 2000 / Accepted: 4 October 2000     

Exclusion of the candidate genes ACE and Bcl-2 for six families with nephronophthisis not linked to the NPH1 locus.

BACKGROUND: Nephronophthisis (NPH) is an autosomal recessively transmitted kidney disease, characterized by cyst formation at the cortico-medullary junction, and a sclerosing tubulointerstitial nephropathy. Juvenile nephronophthisis (NPH1) is the most common genetic cause of renal failure in children and maps to chromosome 2q12-q13. The responsible gene NPHP1 has been identified and encodes for nephrocystin. Not all families with NPH demonstrate linkage to that locus. METHODS: We studied six families with NPH without linkage to the NPH1 locus. In order to attempt identification of a new causative gene, the candidate genes ACE (angiotensin converting enzyme) and Bcl-2 (B cell leukaemia/lymphoma 2 gene) originating from mouse models, were examined. For the six families highly polymorphic microsatellites covering the whole candidate gene regions were haplotyped and linkage analysis was performed. RESULTS: Haplotype analyses of all families examined were incompatible with linkage of the disease status to ACE or Bcl-2. Linkage analysis excluded both candidate gene regions with a LOD-score of < -2. CONCLUSIONS: This study excluded the candidate genes ACE and Bcl-2 for NPH. Additional linkage studies need to be performed in order to identify further genes responsible for nephronophthisis.     

A case report of two Chinese monozygotic twins with NPHP1 gene-associated nephronophthisis undergoing kidney transplantation from a related living-donor

BackgroundNephronophthisis (NPHP) is an autosomal recessive cystic kidney disease and the most common hereditary disease leading to end-stage renal disease in children and adolescents. The NPHP1 gene was the first NPHP gene to be discovered. Pathogenic variation of the NPHP1 gene can cause juvenile renal wasting disease type 1.Case presentationHere, we report the first case of living related kidney transplantation of monozygotic twins with NPHP1 nephronophthisis in China; one of these cases involved cross-blood type kidney transplantation. Our experience shows that patients with NPHP1 nephronophthisis have almost no risk recurrent kidney disease following living related kidney transplantation and genetic testing. The two twins recovered well without any complications.ConclusionsThis is the first report of living related kidney transplantation of monozygotic twins with heterozygous deletion of the NPHP1 gene in a Chinese family with NPHP. In addition, genetic testing provides an efficient means of evaluating the safety of living related kidney transplantation in patients with NPHP1 nephronophthisis.     

Clinical features and mutation of NPHP5 in two Chinese siblings with Senior‐Løken syndrome

Senior‐Løken syndrome is a rare syndromic form of nephronophthisis that is associated with retinal dystrophy. Presently, seven genes (NPHP1‐6 and NPHP10) have been associated with Senior‐Løken syndrome. NPHP5 mutations are known to cause classical Senior‐Løken syndrome. Here, we report two sisters (II‐4, II‐5) from a Chinese Han ethnic family who presented with classical Senior‐Løken syndrome. Both affected sisters exhibited Leber's congenital amaurosis and juvenile nephronophthisis that progressed to end‐stage renal disease by the age of 16 years and 9 months in patient II‐4 and 12 years and 9 months in patient II‐5. Sequence analysis showed a homozygous truncated mutation in NPHP5, c.1090C>T (p.R364X), in the patient II‐4. This mutation is predicted to introduce a new open reading frame that results in the truncation of the C‐terminal 235 amino acids of nephrocystin‐5 and its consequent loss of function. Both parents carried a single heterozygous mutation in the same position, and no homozygous deletion of NPHP1 was found in this pedigree.     

NEK8 mutations affect ciliary and centrosomal localization and may cause nephronophthisis

Nephronophthisis, an autosomal recessive kidney disease, is the most frequent genetic cause of chronic renal failure in the first 3 decades of life. Causative mutations in 8 genes (NPHP1-8) have been identified, and homologous mouse models for NPHP2/INVS and NPHP3 have been described. The jck mouse is another model of recessive cystic kidney disease, and this mouse harbors a missense mutation, G448V, in the highly conserved RCC1 domain of Nek8. We hypothesized that mutations in NEK8 might cause nephronophthisis in humans, so we performed mutational analysis in a worldwide cohort of 588 patients. We identified 3 different amino acid changes that were conserved through evolution (L330F, H425Y, and A497P) and that were absent from at least 80 ethnically matched controls. All 3 mutations were within RCC1 domains, and the mutation H425Y was positioned within the same RCC1 repeat as the mouse jck mutation. To test the functional significance of these mutations, we introduced them into full-length mouse Nek8 GFP-tagged cDNA constructs. We transiently overexpressed the constructs in inner medullary collecting duct cells (IMCD-3 cell line) and compared the subcellular localization of mutant Nek8 to wild-type Nek8. All mutant forms of Nek8 showed defects in ciliary localization to varying degrees; the H431Y mutant (human H425Y) was completely absent from cilia and the amount localized to centrosomes was decreased. Overexpression of these mutants did not affect overall ciliogenesis, mitosis, or centriole number. Our genetic and functional data support the assumption that mutations in NEK8 cause nephronophthisis (NPHP9), adding another link between proteins mutated in cystic kidney disease and their localization to cilia and centrosomes.     

Nephronophthisis-associated ciliopathies

Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, represents the most frequent genetic cause of end-stage kidney disease in the first three decades of life. Contrary to polycystic kidney disease, NPHP shows normal or diminished kidney size, cysts are concentrated at the corticomedullary junction, and tubulointerstitial fibrosis is dominant. NPHP can be associated with retinitis pigmentosa (Senior-L?ken syndrome), liver fibrosis, and cerebellar vermis aplasia (Joubert syndrome) in approximately 10% of patients. Positional cloning of six novel genes (NPHP1 through 6) as mutated in NPHP and functional characterization of their encoded proteins have contributed to the concept of "ciliopathies." It has helped advance a new unifying theory of cystic kidney diseases. This theory states that the products of all genes that are mutated in cystic kidney diseases in humans, mice, or zebrafish are expressed in primary cilia or centrosomes of renal epithelial cells. Primary cilia are sensory organelles that connect mechanosensory, visual, osmotic, and other stimuli to mechanisms of cell-cycle control and epithelial cell polarity. The ciliary theory explains the multiple organ involvement in NPHP regarding retinitis pigmentosa, liver fibrosis, ataxia, situs inversus, and mental retardation. Mutations in NPHP genes cause defects in signaling mechanisms, including the noncanonical Wnt signaling pathway. The "ciliopathy" NPHP thereby is caused by defects in tissue differentiation and maintenance as a result of impaired processing of extracellular cues. Nephrocystins, the proteins that are encoded by NPHP genes, are highly conserved in evolution. Positional cloning of additional causative genes of NPHP will elucidate further signaling mechanisms that are involved, thereby establishing therapeutic approaches using animal models in mouse, zebrafish, and Caenorhabditis elegans.     

Evidence of oligogenic inheritance in nephronophthisis

Nephronophthisis is a recessive cystic renal disease that leads to end-stage renal failure in the first two decades of life. Twenty-five percent of nephronophthisis cases are caused by large homozygous deletions of NPHP1, but six genes responsible for nephronophthisis have been identified. Because oligogenic inheritance has been described for the related Bardet-Biedl syndrome, we evaluated whether mutations in more than one gene may also be detected in cases of nephronophthisis. Because the nephrocystins 1 to 4 are known to interact, we examined patients with nephronophthisis from 94 different families and sequenced all exons of the NPHP1, NPHP2, NPHP3, and NPHP4 genes. In our previous studies involving 44 families, we detected two mutations in one of the NPHP1-4 genes. Here, we detected in six families two mutations in either NPHP1, NPHP3, or NPHP4, and identified a third mutation in one of the other NPHP genes. Furthermore, we found possible digenic disease by detecting one individual who carried one mutation in NPHP2 and a second mutation in NPHP3. Finally, we detected the presence of a single mutation in nine families, suggesting that the second recessive mutation may be in another as yet unidentified NPHP gene. Our findings suggest that oligogenicity may occur in cases of nephronophthisis.     

High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations

Joubert syndrome (JS) is an autosomal recessive disorder that is described in patients with cerebellar ataxia, mental retardation, hypotonia, and neonatal respiratory dysregulation. Kidney involvement (nephronophthisis or cystic renal dysplasia) is associated with JS in one fourth of known cases. Mutations in three genes--AHI1, NPHP1, and NPHP6--have been identified in patients with JS. However, because NPHP1 mutations usually cause isolated nephronophthisis, the factors that predispose to the development of neurologic involvement are poorly understood. In an attempt to identify such genetic determinants, a cohort of 28 families with nephronophthisis and at least one JS-related neurologic symptom were screened for mutations in AHI1, NPHP1, and NPHP6 genes. NPHP1 and NPHP6 homozygous or compound heterozygous mutations were found in 13 (46%) and six (21%) unrelated patients, respectively. Two of the 13 patients with NPHP1 mutations carried either a heterozygous truncating mutation in NPHP6 or a heterozygous missense mutation in AHI1. Furthermore, five patients with NPHP1 mutations carried the AHI1 variant R830W, which was predicted to be "possibly damaging" and was found with significantly higher frequency than in healthy control subjects and in patients with NPHP1 mutations without neurologic symptoms (five of 26 versus four of 276 and three of 152 alleles; P < 0.001 and P < 0.002, respectively). In contrast to the variable neurologic and milder retinal phenotype of patients with NPHP1 mutations, patients with NPHP6 mutations presented with a more severe neurologic and retinal phenotype. In conclusion, NPHP1 and NPHP6 are major genes of nephronophthisis associated with JS. Epistatic effects that are provided by heterozygous NPHP6 and AHI1 mutations and variants may contribute to the appearance of extrarenal symptoms in patients with NPHP1 mutations.     

Nephronophthisis type 1 deletion syndrome with neurological symptoms: prevalence and significance of the association

Type 1 nephronophthisis (NPHP) with homozygous deletions of nephrocystin [NPHP1, DEL] has been considered a pure renal disorder, but co-occurrence of extrarenal symptoms, mainly retinitis pigmentosa, is observed in a subset of patients. Recently, [NPHP1, DEL] has been detected in three patients with Joubert syndrome-related disorders (JSRDs), who associated neurological signs with a peculiar neuroradiological malformation known as the 'molar tooth sign' (MTS). To define the frequency of JSRD spectrum in NPHP1 patients, we re-examined 56 cases with [NPHP1, DEL] and found an overall incidence of 8.9% (five out 56 patients). All had small hyperechoic kidneys and had developed advanced renal failure within 15 years. Two patients presented the complete features of JSRD with cerebello-renal-retinal association and MTS. Two others showed, instead, severe intentional tremor and thick superior cerebellar peduncles on brain magnetic resonance imaging (MRI), and one of them had associated retinopathy. The fifth patient presented with hypotonia, developmental delay, central deafness, and ataxia associated with Leber congenital amaurosis and liver fibrosis but with normal brain MRI. Marked intrafamilial variability of associated extrarenal symptoms was observed in familial cases. Deletion extension did not differ in patients with isolated renal phenotype and in those with associated neurological symptoms. In conclusion, neurological defects varying from subtle involvement of cerebellum with thickened peduncle to both JSRD and diffuse central hypotonia are frequent in [NPHP1, DEL] patients. Prevalence of such association may justify systematic neurological and neuroradiological evaluation.     

Evidence for further genetic heterogeneity in nephronophthisis.

BACKGROUND: A new type of nephronophthisis (NPH) has been recently identified in a large Venezuelan kindred: adolescent nephronophthisis (NPH3) causes end-stage renal disease (ESRD) at a median age of 19 years. The responsible gene (NPHP3) maps to 3q21-q22. NPH3 shares with juvenile nephronophthisis (NPH1) the same disease manifestations such as polyuria, polydipsia, and secondary enuresis. Histopathological findings consist of tubular basement membrane changes, cysts at the corticomedullary junction, and a chronic sclerosing tubulointerstitial nephropathy. The only difference is a younger age at ESRD in NPH1 (median age of 13 years) when compared with NPH3. METHODS: In order to evaluate whether there might be a fourth locus of isolated nephronophthisis, we studied eight NPH families without extrarenal disease manifestations and without linkage to the NPH1 locus (NPHP1) on chromosome 2q12-q13. ESRD was reached at ages ranging from 7 to 33 years. Individuals were haplotyped with microsatellites covering the genetic locus of NPHP3. Infantile NPH (NPH2) was excluded in all families by the clinical history and histological findings. RESULTS: In four of the examined families haplotype analysis was compatible with linkage to the NPHP3 locus. In one of these families identity by descent was observed. In contrast, in another four families linkage was excluded for NPHP3. CONCLUSION: Four NPH-families were neither linked to NPHP1 nor to NPHP3, indicating further genetic heterogeneity within the group of nephronophthisis. The finding of further genetic heterogeneity in NPH has important implications for genetic counselling.     

Pseudodominant inheritance of nephronophthisis caused by a homozygous NPHP1 deletion

Nephronophthisis (NPHP) is an autosomal recessive kidney disease characterized by tubular basement membrane disruption, interstitial infiltration, and tubular cysts. NPHP leads to end-stage renal failure (ESRD) in the first three decades of life and is the most frequent genetic cause of chronic renal failure in children and young adults. Extrarenal manifestations are known, such as retinitis pigmentosa, brainstem and cerebellar anomalies, liver fibrosis, and ocular motor apraxia type Cogan. We report on a Turkish family with clinical signs of nephronophthisis. The phenotype occurred in two generations and therefore seemed to be inherited in an autosomal dominant pattern. Nevertheless, a deletion analysis of the NPHP1 gene on chromosome 2 was performed and showed a homozygous deletion. Analysis of the family pedigree indicated no obvious consanguinity in the last three generations. However, haplotype analysis demonstrated homozygosity on chromosome 2 indicating a common ancestor to the parents of all affected individuals. NPHP1 deletion analysis should always be considered in patients with apparently dominant nephronophthisis. Furthermore, three out of four patients developed ESRD between 27 and 43 years of age, which may be influenced by yet unknown modifier genes.     

Establishing an algorithm for molecular genetic diagnostics in 127 families with juvenile nephronophthisis

BACKGROUND: Juvenile nephronophthisis (NPH1), an autosomal recessive cystic disease of the kidney, represents the most common genetic cause of end-stage renal disease in the first two decades of life. On the basis of identification of the gene (NPHP1) defective in NPH1 and the presence of homozygous deletions of NPHP1 in the majority of NPH1 patients, molecular genetic diagnosis for NPH1 is now possible. Molecular genetic testing offers the only method for definite diagnosis of NPH1 and avoids invasive diagnostic measures like renal biopsy. METHODS: We examined 127 families (204 patients) with the presumed diagnosis of NPH using molecular genetic diagnostic techniques. In 68 families, renal biopsy was performed and was consistent with NPH, and in 61 families, there was more than one affected child ("multiplex families"). RESULTS: In 74 families (115 patients), there was proof of the diagnosis of NPH1 by detection of a homozygous deletion of the NPHP1 gene, and in 5 families a heterozygous deletion in combination with a point mutation in NPHP1 was demonstrated. Furthermore, for 16 families, NPH1 was excluded with high likelihood by linkage analysis, and for 20 families by detection of heterozygosity for two newly identified polymorphic markers within the deletion region. In 5 of the remaining 12 families, which were noninformative for these markers, fluorescence in situ hybridization did not detect any further heterozygous deletions. CONCLUSIONS: The diagnosis of NPH1 was proven by molecular genetic techniques in 62% of families with one or more children with the presumed diagnosis of NPH. We present evidence that there is a fourth locus for NPH, since only 6 of the 26 multiplex families in whom the diagnosis of NPH1 was excluded were compatible with linkage to other loci for NPH. On the basis of the presented data, we propose an algorithm for molecular genetic diagnostics in NPH.