Novel NPHS2 variant in patients with familial steroid-resistant nephrotic syndrome with early onset, slow progression and dominant inheritance pattern.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is a common cause of end-stage renal disease in children but also occurs as an adult-onset condition. In a subset of SRNS patients, pathogenic variants are found in genes coding for podocyte foot process proteins. The aim of this study was to define the role of pathogenic variants in Finnish patients with familial and sporadic SRNS. METHODS: We analyzed SRNS-related genes NPHS1, NPHS2, NEPH1, ACTN4, TRPC6, INF2, WT1, CD2AP, LAMB2, and PLCE1 for disease-causing variants using direct sequencing of exons and intron/exon boundaries in all members of a family with dominant SRNS with early onset and slow progression to end-stage renal disease. We carried out a whole genome sequencing in two affected and two healthy family members. The function of found podocin variant was studied using co-immunoprecipitation and immunohistochemistry. Podocyte gene sequences were analyzed in a cohort of Finnish non-familial SRNS patients. RESULTS: A heterozygous de novo deletion, c.988_989delCT in NPHS2, was found in all affected family members and in none of their healthy relatives, non-familial patients or controls. No other SRNS-related gene variant, coding or non-coding co-segregated with the disease phenotype in the family. While the truncated podocin remained able to bind nephrin, the expression of nephrin was fragmented and podocin expression reduced. The gene analysis of the non-familial SRNS patients revealed few variants. CONCLUSION: The role of podocin variants in nephrotic syndrome may be more varied than previously thought.

Single Nucleotide Polymorphisms in Pediatric Idiopathic Nephrotic Syndrome.

Polymorphic variants in several molecules involved in the glomerular function and drug metabolism have been implicated in the pathophysiology of pediatric idiopathic nephrotic syndrome (INS), but the results remain inconsistent. We analyzed the association of eleven allelic variants in eight genes (angiopoietin-like 4 (ANGPTL4), glypican 5 (GPC5), interleukin-13 (IL-13), macrophage migration inhibitory factor (MIF), neural nitric oxide synthetase (nNOS), multidrug resistance-1 (MDR1), glucocorticoid-induced transcript-1 (GLCCI1), and nuclear receptor subfamily-3 (NR3C1)) in 100 INS patients followed up till adulthood. We genotyped variants using PCR and direct sequencing and evaluated estimated haplotypes of MDR1 variants. The analysis revealed few differences in SNP genotype frequencies between patients and controls, or in clinical parameters among the patients. Genotype distribution of MDR1 SNPs rs1236, rs2677, and rs3435 showed significant (p < 0.05) association with different medication regimes (glucocorticoids only versus glucocorticoids plus additional immunosuppressives). Some marginal association was detected between ANGPTL4, GPC5, GLCCI1, and NR3C1 variants and different medication regimes, number of relapses, and age of onset. Conclusion. While MDR1 variant genotype distribution associated with different medication regimes, the other analyzed gene variants showed only little or marginal clinical relevance in INS.

Podocyte proteins in congenital and minimal change nephrotic syndrome.

BACKGROUND: Podocyte foot process effacement is a uniform finding in kidneys with heavy proteinuria. Its molecular mechanisms, however, are unsolved. We analyzed the expression of podocyte proteins in two kidney disorders: Congenital nephrotic syndrome of the Finnish type (CNF) and minimal change nephrotic syndrome (MCNS). METHODS: Immunoperoxidase and immunofluorescence stainings were used to semiquantitatively analyze the expression of 13 and 4 podocyte proteins from different cellular compartments in CNF and MCNS, respectively. RESULTS: The expression of a major slit diaphragm (SD) protein, Neph 1, showed a 46-fold decrease (p < 0.0001) in CNF kidneys as compared to controls. The three cytosolic adaptor proteins, podocin, NCK1/2, CD2AP, connecting SD proteins to the actin cytoskeleton were slightly upregulated (1.1-fold, 1.4-fold, and 3.3-fold, respectively). Also, the staining of the two actin-regulator proteins, ACTN4 and INF2, was modestly increased (2.2-fold and 1.7-fold, respectively, p < 0.0001). Staining for α3-integrin showed 1.9-fold increase (p < 0.0001) indicating that the major podocyte anchoring complex, α3ß1, was well preserved in CNF glomeruli. In contrast to CNF kidneys, Neph1 FAT1, ACTN4, and CD2AP were quite normally expressed in proteinuric and non-proteinuric MCNS kidneys. CONCLUSION: CNF kidneys lacking nephrin show decreased expression of other SD proteins but not cytosolic podocyte proteins involved in the foot process architecture or function. In MCNS kidneys, these changes in expression were not observed.

The Finnish disease heritage database (FinDis) update-a database for the genes mutated in the Finnish disease heritage brought to the next-generation sequencing era.

The Finnish Disease Heritage Database (FinDis) (http://findis.org) was originally published in 2004 as a centralized information resource for rare monogenic diseases enriched in the Finnish population. The FinDis database originally contained 405 causative variants for 30 diseases. At the time, the FinDis database was a comprehensive collection of data, but since 1994, a large amount of new information has emerged, making the necessity to update the database evident. We collected information and updated the database to contain genes and causative variants for 35 diseases, including six more genes and more than 1,400 additional disease-causing variants. Information for causative variants for each gene is collected under the LOVD 3.0 platform, enabling easy updating. The FinDis portal provides a centralized resource and user interface to link information on each disease and gene with variant data in the LOVD 3.0 platform. The software written to achieve this has been open-sourced and made available on GitHub (http://github.com/findis-db), allowing biomedical institutions in other countries to present their national data in a similar way, and to both contribute to, and benefit from, standardized variation data. The updated FinDis portal provides a unique resource to assist patient diagnosis, research, and the development of new cures.

[Finnish disease heritage]. / Suomalainen tautiperintö.

The Finnish disease heritage refers to rare hereditary diseases that occur in the Finnish population in a relatively larger proportion than in other populations. The genes underlying all of the 36 diseases of the disease heritage have been identified. Together with her group and collaborators, Leena Palotie identified 15 of these, and this review includes the description of some of these achievements. As a result of the so-called founder effect, one predominant mutation underlying these diseases occurs in our population, facilitating the diagnostics of these diseases in our country.

Mutant CHUK and severe fetal encasement malformation.

We report an autosomal recessive lethal syndrome characterized by multiple fetal malformations, the most obvious anomalies being the defective face and seemingly absent limbs, which are bound to the trunk and encased under the skin. We identified the molecular defect that causes this syndrome, using a combined strategy of gene-expression arrays, candidate-gene analysis, clinical studies, and genealogic investigations. A point mutation in two affected fetuses led to the loss of the conserved helix­loop­helix ubiquitous kinase (CHUK), also known as IκB kinase α. CHUK has an essential role in the development of skin epidermis and its derivatives, along with various other morphogenetic events. (Funded by the Academy of Finland and others.).

Mutation spectrum of Meckel syndrome genes: one group of syndromes or several distinct groups?

Meckel syndrome (MKS) is a lethal malformation syndrome that belongs to the group of disorders that are associated with primary cilia dysfunction. Total of five genes are known to be involved in the molecular background of MKS. Here we have systematically analyzed all these genes in a total of 29 MKS families. Seven of the families were Finnish and the rest originated from elsewhere in Europe. We found 12 novel mutations in 13 families. Mutations in the MKS genes are also found in other syndromes and it seems reasonable to assume that there is a correlation between the syndromes and the mutations. To obtain some supportive information, we collected all the previously published mutations in the genes to see whether the different syndromes are dictated by the nature of the mutations. Based on this study, mutations play a role in the clinical phenotype, given that the same allelic combination of mutations has never been reported in two clinically distinct syndromes.

Unraveling the disease pathogenesis behind lethal hydrolethalus syndrome revealed multiple changes in molecular and cellular level.

BACKGROUND: Hydrolethalus syndrome (HLS) is a severe fetal malformation syndrome characterized by multiple developmental anomalies, including central nervous system (CNS) malformation such as hydrocephaly and absent midline structures of the brain, micrognathia, defective lobation of the lungs and polydactyly. Microscopically, immature cerebral cortex, abnormalities in radial glial cells and hypothalamic hamartoma are among key findings in the CNS of HLS fetuses. HLS is caused by a substitution of aspartic acid by glycine in the HYLS1 protein, whose function was previously unknown. RESULTS: To provide insight into the disease mechanism(s) of this lethal disorder we have studied different aspects of HLS and HYLS1. A genome-wide gene expression analysis indicated several upregulated genes in cell cycle regulatory cascades and in specific signal transduction pathways while many downregulated genes were associated with lipid metabolism. These changes were supported by findings in functional cell biology studies, which revealed an increased cell cycle rate and a decreased amount of apoptosis in HLS neuronal progenitor cells. Also, changes in lipid metabolism gene expression were reflected by a significant increase in the cholesterol levels of HLS liver tissues. In addition, based on our functional studies of HYLS1, we propose that HYLS1 is a transcriptional regulator that shuffles between the cytoplasm and the nucleus, and that when HYLS1 is mutated its function is significantly altered. CONCLUSION: In this study, we have shown that the HYLS1 mutation has significant consequences in the cellular and tissue levels in HLS fetuses. Based on these results, it can be suggested that HYLS1 is part of the cellular transcriptional regulatory machinery and that the genetic defect has a widespread effect during embryonic and fetal development. These findings add a significant amount of new information to the pathogenesis of HLS and strongly suggest an essential role for HYLS1 in normal fetal development.

The mutation spectrum in RECQL4 diseases.

Mutations in the RECQL4 gene can lead to three clinical phenotypes with overlapping features. All these syndromes, Rothmund-Thomson (RTS), RAPADILINO and Baller-Gerold (BGS), are characterized by growth retardation and radial defects, but RAPADILINO syndrome lacks the main dermal manifestation, poikiloderma that is a hallmark feature in both RTS and BGS. It has been previously shown that RTS patients with RECQL4 mutations are at increased risk of osteosarcoma, but the precise incidence of cancer in RAPADILINO and BGS has not been determined. Here, we report that RAPADILINO patients identified as carriers of the c.1390+2delT mutation (p.Ala420_Ala463del) are at increased risk to develop lymphoma or osteosarcoma (6 out of 15 patients). We also summarize all the published RECQL4 mutations and their associated cancer cases and provide an update of 14 novel RECQL4 mutations with accompanying clinical data.

Hydrolethalus syndrome: neuropathology of 21 cases confirmed by HYLS1 gene mutation analysis.

Hydrolethalus syndrome is a lethal malformation syndrome with a severe brain malformation, most often hydrocephaly and absent midline structures. Other frequent findings are micrognathia, polydactyly, and defective lobation of the lungs. Hydrolethalus syndrome is inherited in an autosomal recessive manner and is caused by a missense mutation in the HYLS1 gene. Here, we report the neuropathologic features of 21 genetically confirmed cases. Typically, 2 separated cerebral hemispheres could be identified, but they lacked midline and olfactory structures and were situated basally with a massive accumulation of cerebrospinal fluid. Temporal and occipital lobes were hypoplastic, and normally developed hippocampi were not found. Primitive thalami and basal ganglia were fused in the midline. A hypothalamic hamartoma was a frequent finding, and brainstem and cerebellum were hypoplastic. Three cases were hydranencephalic, and 1 was anencephalic. A midline "keyhole" defect in the skull base was a constant finding. Histologically, the cortex was dysplastic. This pattern of brain pathology, clearly belonging to the midline patterning defects, seems to be unique for the hydrolethalus syndrome and combines features of disturbed neurulation, prosencephalization, and migration. Despite variation in the clinicopathologic phenotype, all cases in the series carried the same homozygous missense mutation in HYLS1.

Identification of CC2D2A as a Meckel syndrome gene adds an important piece to the ciliopathy puzzle.

Meckel syndrome (MKS) is a lethal malformation disorder characterized classically by encephalocele, polycystic kidneys, and polydactyly. MKS is also one of the major contributors to syndromic neural tube defects (NTDs). Recent findings have shown primary cilia dysfunction in the molecular background of MKS, indicating that cilia are critical for early human development. However, even though four genes behind MKS have been identified to date, they elucidate only a minor proportion of the MKS cases. In this study, instead of traditional linkage analysis, we selected 10 nonrelated affected fetuses and looked for the homozygous regions shared by them. Based on this strategy, we identified the sixth locus and the fifth gene, CC2D2A (MKS6), behind MKS. The biological function of CC2D2A is uncharacterized, but the corresponding polypeptide is predicted to be involved in ciliary functions and it has a calcium binding domain (C2). Immunofluorescence staining of patient's fibroblast cells demonstrates that the cells lack cilia, providing evidence for the critical role of CC2D2A in cilia formation. Our finding is very significant not only to understand the molecular background of MKS, but also to obtain additional information about the function of the cilia, which can help to understand their significance in normal development and also in other ciliopathies, which are an increasing group of disorders with overlapping phenotypes.

Mutations in mRNA export mediator GLE1 result in a fetal motoneuron disease.

The most severe forms of motoneuron disease manifest in utero are characterized by marked atrophy of spinal cord motoneurons and fetal immobility. Here, we report that the defective gene underlying lethal motoneuron syndrome LCCS1 is the mRNA export mediator GLE1. Our finding of mutated GLE1 exposes a common pathway connecting the genes implicated in LCCS1, LCCS2 and LCCS3 and elucidates mRNA processing as a critical molecular mechanism in motoneuron development and maturation.

Atypical Rothmund-Thomson syndrome in a patient with compound heterozygous mutations in RECQL4 gene and phenotypic features in RECQL4 syndromes.

We describe the natural history of the RTSII phenotype in a 7-year-old boy who developed intrauterine and postnatal growth retardation, failure to thrive and persisting diarrhoea. The growth hormone stimulation test identified an isolated growth hormone deficiency. Since infancy, the patient manifested skin lesions characterized by a very mild poikilodermic-like appearance on the cheeks only, widespread café-au-lait spots and the absence of eyebrows and eyelashes. There was no cataract. Orthopaedic and radiologic work-up identified the absence of thumb anomaly and radial head luxation and patellar hypoplasia. Neurologic, cognitive milestones and intelligence were normal. The cytogenetic work-up did not show any anomaly. Based on this clinical presentation, we carried out a sequencing analysis of the RECQL4 gene, which is responsible for Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes and found a splice site mutation (IVS10-1G>A) and a nucleotide substitution in exon 12 (L638P). The mother was identified as a carrier for the substitution in exon 12 and the father for the splice site mutation, respectively. An analysis of the transcripts focused on the RECQL4 helicase domain: in the proband only those generated from the maternal L638 allele were present. This case report emphasizes the clinical overlap between RAPADILINO and Rothmund-Thomson syndromes within a continuum phenotypic spectrum. The distinctive set of clinical signs displayed by the patient may be accounted for by his unique combination of two different RECQL4 mutations. The molecular findings provide information that enhances our comprehension of genotype-phenotype correlations in RECQL4 diseases, enables a more precise genetic counseling to the parents and facilitates a more appropriate long-term follow-up to the affected child.

Misleading findings of homozygosity mapping resulting from three novel mutations in NPHS1 encoding nephrin in a highly inbred community.

PURPOSE: Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) is a rare autosomal recessive disease caused by mutations in the NPHS1 gene encoding nephrin. We diagnosed congenital nephrotic syndrome in 12 children living in a village near Jerusalem. Most of the inhabitants are descendants of one Muslim family and have maintained their isolation by preference of consanguineous marriages. The aim of this study was to confirm that the NPHS1 gene is responsible for congenital nephrotic syndrome in our population, applying homozygosity mapping. METHODS: DNA samples were genotyped by four microsatellite markers that were in linkage disequilibrium with the NPHS1 gene on chromosome 19q13.1. Immunoperoxidase staining was used to study the expression of nephrin, and mutations were subsequently identified by direct sequencing of the entire coding region of the NPHS1 gene. RESULTS: Haplotype analysis revealed several different haplotypes, leading us to assume erroneously that there was genetic heterogeneity of congenital nephrotic syndrome. Because nephrin was completely absent in kidney tissue of one patient, direct sequencing of all DNA samples was performed, yielding three novel mutations: c.1138C>T (p.Gln380X), c.2160_ 2161insC (p.Cys721fs), and c.1707C>G (p.Ser569Arg). Patients were either homozygous for one of these mutations or compound heterozygotes, and they differed in their phenotype. CONCLUSION: We report the potential pitfalls of performing homozygosity mapping in a highly consanguineous population and discuss the phenomenon of multiple mutations in a given gene within an isolate.

Neural precursor cells from a fatal human motoneuron disease differentiate despite aberrant gene expression.

Precursor cells of the human central nervous system can be cultured in vitro to reveal pathogenesis of diseases or developmental disorders. Here, we have studied the biology of neural precursor cells (NPCs) from patients of lethal congenital contracture syndrome (LCCS), a severe motoneuron disease leading to prenatal death before the 32nd gestational week. LCCS fetuses are immobile because of a motoneuron defect, seen as degeneration of the anterior horn and descending tracts of the developing spinal cord. The genetic defect for the syndrome is unknown. We show that NPCs isolated postmortem from LCCS fetuses grow and are maintained in culture, but display increased cell cycle activity. Global transcript analysis of undifferentiated LCCS precursor cells present with changes in EGF-related signaling when compared with healthy age-matched human controls. Further, we show that LCCS-derived NPCs differentiate into cells of neuronal and glial lineage and that the initial differentiation is not accompanied by overt apoptosis. Cells expressing markers Islet-1 and Hb9 are also generated from the LCCS NPCs, suggesting that the pathogenic mechanism of LCCS does not directly affect the differentiation capacity or survival of the cells, but the absence of motoneurons in LCCS may be caused by a noncell autonomous mechanism.

Lethal congenital contracture syndrome (LCCS) and other lethal arthrogryposes in Finland--an epidemiological study.

Arthrogryposis multiplex congenita is a heterogeneous group of disorders characterized by multiple contractures with an estimated frequency of 1 in 3,000 births. With improving diagnostic methods, increasing numbers of fetuses with arthrogryposis are found. The pathogenetic mechanisms are relatively well known but the epidemiology and genetics of the prenatally lethal forms of arthrogryposis are less well known. In this study we collected all cases of a multiple contractures diagnosed in Finland during 1987-2002 including live born infants, stillbirths, and terminated pregnancies. Ninety-two cases of 214 suffered intrauterine demise (68 selective pregnancy terminations and 24 stillbirths) and 58 died in infancy. In 141 out of these cases the diagnosis could be included within lethal arthrogryposes, with a prevalence of 1 in 6,985 (1.43/10,000) births. Of these, 59 had spinal cord pathology at autopsy and thus were of neurogenic origin. Thirty-nine cases had lethal congenital contracture syndrome (LCCS) clinically characterized by total immobility of the fetus at all ultrasound examinations (12 weeks or later), multiple joint contractures in both upper and lower limbs, hydrops, and fetal death before the 32nd week of pregnancy. LCCS is noted as a unique Finnish disorder with a prevalence of 1 in 25,250 (0.40/10,000) births and is a major cause of lethal arthrogryposis in Finland.

Recurrence of proteinuria following renal transplantation in congenital nephrotic syndrome of the Finnish type.

We report a Caucasian boy of Italian descent with congenital nephrotic syndrome of the Finnish type (NPHS1, CNF, MIM 256300) who developed recurrence of proteinuria and hypoalbuminemia on the seventh post-operative day following living related renal transplantation from his paternal aunt. The allograft biopsy was normal except for effacement of podocyte foot processes on electron microscopy. He was treated by the substitution of mycophenolate mofetil with cyclophosphamide for 12 weeks, in addition to cyclosporine, prednisone and daclizumab. His proteinuria resolved quickly following the initiation of cyclophosphamide treatment, and he remains in remission 4 years after receiving his transplant. His native and allograft kidneys were evaluated for nephrin expression by immunohistochemistry, DNA analysis for the NPHS1 mutation, serum for the presence of auto-antibodies to nephrin by both enzyme-linked immunosorbent assay (ELISA) and fetal glomeruli immunofluorescence assay, and serum for glomerular permeability to albumin (Palb) activity using a functional in vitro assay for Palb. Nephrin expression was completely absent in the native kidney, while it was decreased in the allograft compared with normal. DNA analysis of the NPHS1 gene revealed mutations 3248G>T and 3250delG in exon 24, causing G1083V and 1084Vfs, respectively, inherited from his father, and 3478C>T in exon 27, that leads to R1160X, inherited from his mother. Serum was negative for auto-antibodies to nephrin. Interestingly, the Palb activity was increased at the time of recurrence of proteinuria following transplantation (Palb 0.73+/-0.10) and remained elevated when retested more than 3 years later (Palb 0.54+/-0.09). This is the first report of increased Palb activity in recurrence of proteinuria following transplantation in NPHS1. We speculate the role of increased Palb activity in the recurrence of proteinuria following transplantation in NPHS1.

MKS1, encoding a component of the flagellar apparatus basal body proteome, is mutated in Meckel syndrome.

Meckel syndrome (MKS) is a severe fetal developmental disorder reported in most populations. The clinical hallmarks are occipital meningoencephalocele, cystic kidney dysplasia, fibrotic changes of the liver and polydactyly. Here we report the identification of a gene, MKS1, mutated in MKS families linked to 17q. Mks1 expression in mouse embryos, as determined by in situ hybridization, agrees well with the tissue phenotype of MKS. Comparative genomics and proteomics data implicate MKS1 in ciliary functions.

Indicative oligodendrocyte dysfunction in spinal cords of human fetuses suffering from a lethal motoneuron disease.

Human spinal cord development is still poorly understood and detailed molecular analyses of human motoneuron diseases could improve our understanding of the normal developmental processes of the spinal cord. Lethal Congenital Contracture Syndrome (LCCS, MIM 253310) provides a human model to study the early motoneuronal development. A typical phenotype of LCCS fetuses consists of multiple joint contractures, distinct facial features, and hydrops. Tissue pathology is characterized by severe muscle atrophy, lung hypoplasia, and degeneration of the anterior horn of the spinal cord as the hallmark of the syndrome. In this study we performed a global transcript analysis of LCCS spinal cords. The RNA expression profiles of these spinal cords were compared to age-matched healthy control fetuses, aborted for nonrelated causes. In addition, we applied phylogenetic footprinting methods to decipher the mechanisms of transcriptional regulation in the affected transcripts. Changes in transcripts involved with the development of the CNS and oligodendrocytes were obvious and the transcription factor PAX6 was identified as a key regulator during spinal cord development. In addition, transcript pathway analysis clearly indicated genes belonging to groups with neuronal functions to be affected. Our findings support the hypothesis that human motoneurons and oligodendrocytes are dependent on each other during their development and are influenced by distinct transcription factors previously known to act during murine and chick motoneuron development. These data provide valuable information about the molecular pathways putatively active in motoneuron diseases.

Clinical features and outcome of childhood minimal change nephrotic syndrome: is genetics involved?

The pathogenesis of minimal change nephrotic syndrome (MCNS) is still unknown. We performed a clinical and genetic evaluation of 104 adults (mean age 35 years) who presented with MCNS in childhood (mean follow-up 30 years). Clinical data and the present health status were evaluated. Also, the genes encoding the four major slit diaphragm proteins, nephrin, podocin, Neph1 and CD2-associated protein were sequenced in 38 patients with MCNS of varying severity. MCNS presented at the mean age of 5 years, and 80% of the patients relapsed 1-28 (median 3) times during childhood. The 14 subjects (14%) who had proteinuric episodes still in adulthood had a refractory disease already as children. The participants did not show a strong tendency for allergy or immune diseases, and no familial clustering of MCNS was observed. The genetic analyses revealed heterozygous amino acid changes in nephrin and podocin in 10 of the 38 patients studied. On the other hand, the genes coding for Neph1 and CD2AP were highly conserved and no amino acid substitutions were detected. In conclusion, MCNS is a multifactorial disease, in which genetics play a minor role. Allelic variants of the podocyte proteins may, however, modify the phenotype in occasional individuals.