Molecular and cellular pathogenesis of autosomal recessive polycystic kidney disease

Autosomal recessive polycystic kidney disease (ARPKD) is an inherited disease characterized by a malformation complex which includes cystically dilated tubules in the kidneys and ductal plate malformation in the liver. The disorder is observed primarily in infancy and childhood, being responsible for significant pediatric morbidity and mortality. All typical forms of ARPKD are caused by mutations in a single gene, PKHD1 (polycystic kidney and hepatic disease 1). This gene has a minimum of 86 exons, assembled into multiple differentially spliced transcripts and has its highest level of expression in kidney, pancreas and liver. Mutational analyses revealed that all patients with both mutations associated with truncation of the longest open reading frame-encoded protein displayed the severe phenotype. This product, polyductin, is a 4,074-amino acid protein expressed in the cytoplasm, plasma membrane and primary apical cilia, a structure that has been implicated in the pathogenesis of different polycystic kidney diseases. In fact, cholangiocytes isolated from an ARPKD rat model develop shorter and dysmorphic cilia, suggesting polyductin to be important for normal ciliary morphology. Polyductin seems also to participate in tubule morphogenesis and cell mitotic orientation along the tubular axis. The recent advances in the understanding of in vitro and animal models of polycystic kidney diseases have shed light on the molecular and cellular mechanisms of cyst formation and progression, allowing the initiation of therapeutic strategy designing and promising perspectives for ARPKD patients. It is notable that vasopressin V2 receptor antagonists can inhibit/halt the renal cystic disease progression in an orthologous rat model of human ARPKD.

Hydronephrosis in Schinzel-Giedion syndrome: an important clue for the diagnosis

Schinzel-Giedion syndrome is a rare autosomal recessive disorder characterized by coarse facies, midface retraction, hypertrichosis, multiple skeletal anomalies, and cardiac and renal malformations. Craniofacial abnormalities of this syndrome sometimes resemble a storage or metabolic disease. The pathogenesis of the disease remains unknown. The objective of this report was to emphasize the importance of congenital bilateral hydronephrosis for the diagnosis of Schinzel-Giedion syndrome. We describe the first Brazilian case of a newborn with typical facies, generalized hypertrichosis, cardiac and skeletal anomalies, and bilateral hydronephrosis detected during pregnancy and confirmed later by abdominal ultrasonography. Chromosomal constitution was normal. Of the 35 cases already reported in the literature, 31 presented hydronephrosis, which is considered an important clue in diagnosis. If Schinzel-Giedion syndrome were indexed as a cause of congenital hydronephrosis, its identification would be greatly facilitated, since the majority of the other findings in Schinzel-Giedion syndrome are nonspecific and common to many genetic syndromes.

Prevalence study of SNP c.421G>T in the ADAMTS2 gene responsible for dermatosparaxis in White Dorper sheep in Brazil

Abstract: Dermatosparaxis is an autosomal recessive disorder of connective tissue; the disorder is clinically characterized by skin fragility and hyperextensibility. Dermatosparaxis in White Dorper sheep is caused by a single nucleotide polymorphism (SNP) (c.421G>T) in the ADAM metalloproteinase with thrombospondin type 1 motif, 2 (ADAMTS2) gene. The aim of this study was to investigate the prevalence of this SNP in a White Dorper herd in São Paulo state, Brazil. In this study, we collected blood DNA samples from 303 White Dorper sheep and performed polymerase chain reaction to amplify the SNP region. The samples were sequenced to determine the presence of the SNP in the ADAMTS2 gene. The SNP prevalence in the studied population was 15.5%; this finding indicates that more effective control measures should be used to prevent the inheritance of SNP c.421G>T in the ADAMTS2 gene in Brazilian White Dorper herds.

Diagnosis of Smith-Lemli-Opitz syndrome by ultraviolet spectrophotometry

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder due to an inborn error of cholesterol metabolism, characterized by congenital malformations, dysmorphism of multiple organs, mental retardation and delayed neuropsychomotor development resulting from cholesterol biosynthesis deficiency. A defect in 3ß-hydroxysteroid-delta7-reductase (delta7-sterol-reductase), responsible for the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol, causes an increase in 7-DHC and frequently reduces plasma cholesterol levels. The clinical diagnosis of SLOS cannot always be conclusive because of the remarkable variability of clinical expression of the disorder. Thus, confirmation by the measurement of plasma 7-DHC levels is needed. In the present study, we used a simple, fast, and selective method based on ultraviolet spectrophotometry to measure 7-DHC in order to diagnose SLOS. 7-DHC was extracted serially from 200 µl plasma with ethanol and n-hexane and the absorbance at 234 and 282 nm was determined. The method was applied to negative control plasma samples from 23 normal individuals and from 6 cases of suspected SLOS. The method was adequate and reliable and 2 SLOS cases were diagnosed.

Transient high-level expression of ß-galactosidase after transfection of fibroblasts from GM1 gangliosidosis patients with plasmid DNA

GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1) to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 µL lipofectamine 2000 and 1.5-2.0 µg DNA. Cells (2 x 10(5)/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß-Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein) compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients' skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.

Griscelli syndrome-type 2 in twin siblings: case report and update on RAB27A human mutations and gene structure

Griscelli syndrome (GS) is a rare autosomal recessive disorder caused by mutation in the MYO5A (GS1, Elejalde), RAB27A (GS2) or MLPH (GS3) genes. Typical features of all three subtypes of this disease include pigmentary dilution of the hair and skin and silvery-gray hair. Whereas the GS3 phenotype is restricted to the pigmentation dysfunction, GS1 patients also show primary neurological impairment and GS2 patients have severe immunological deficiencies that lead to recurrent infections and hemophagocytic syndrome. We report here the diagnosis of GS2 in 3-year-old twin siblings, with silvery-gray hair, immunodeficiency, hepatosplenomegaly and secondary severe neurological symptoms that culminated in multiple organ failure and death. Light microscopy examination of the hair showed large, irregular clumps of pigments characteristic of GS. A homozygous nonsense mutation, C-T transition (c.550C>T), in the coding region of the RAB27A gene, which leads to a premature stop codon and prediction of a truncated protein (R184X), was found. In patient mononuclear cells, RAB27A mRNA levels were the same as in cells from the parents, but no protein was detected. In addition to the case report, we also present an updated summary on the exon/intron organization of the human RAB27A gene, a literature review of GS2 cases, and a complete list of the human mutations currently reported in this gene. Finally, we propose a flow chart to guide the early diagnosis of the GS subtypes and Chédiak-Higashi syndrome.

Friedreich's ataxia: clinical and molecular study of 25 Brazilian cases

INTRODUCTION: Friedreich's ataxia is a neurodegenerative disorder whose clinical diagnostic criteria for typical cases basically include: a) early age of onset (< 20 or 25 years), b) autosomal recessive inheritance, c) progressive ataxia of limbs and gait, and d) absence of lower limb tendon reflexes. METHODS: We studied the frequency and the size of expanded GAA and their influence on neurologic findings, age at onset, and disease progression in 25 Brazilian patients with clinical diagnosis of Friedreich's ataxia - 19 typical and 6 atypical - using a long-range PCR test. RESULTS: Abnormalities in cerebellar signs, in electrocardiography, and pes cavus occurred more frequently in typical cases; however, plantar response and speech were more frequently normal in this group when the both typical and atypical cases were compared. Homozygous GAA expansion repeats were detected in 17 cases (68%) - all typical cases. In 8 patients (32%) (6 atypical and 2 typical), no expansion was observed, ruling out the diagnosis of Friedreich's ataxia. In cases with GAA expansions, foot deformity, cardiac abnormalities, and some neurologic findings occurred more frequently; however, abnormalities in cranial nerves and in tomographic findings were detected less frequently than in patients without GAA expansions. DISCUSSION: Molecular analysis was imperative for the diagnosis of Friedreich's ataxia, not only for typical cases but also for atypical ones. There was no genotype-phenotype correlation. Diagnosis based only on clinical findings is limited; however, it aids in better screening for suspected cases that should be tested. Evaluation for vitamin E deficiency is recommended, especially in cases without GAA expansion.

The expression of the ACTH receptor

Adrenal glucocorticoid secretion is regulated by adrenocorticotropic hormone (ACTH) acting through a specific cell membrane receptor (ACTH-R). The ACTH-R is a member of the G protein superfamily-coupled receptors and belongs to the subfamily of melanocortin receptors. The ACTH-R is mainly expressed in the adrenocortical cells showing a restricted tissue specificity, although ACTH is recognized by the other four melanocortin receptors. The cloning of the ACTH-R was followed by the study of this gene in human diseases such as familial glucocorticoid deficiency (FGD) and adrenocortical tumors. FGD is a rare autosomal recessive disease characterized by glucocorticoid deficiency, elevated plasma ACTH levels and preserved renin/aldosterone secretion. This disorder has been ascribed to an impaired adrenal responsiveness to ACTH due to a defective ACTH-R, a defect in intracellular signal transduction or an abnormality in adrenal cortical development. Mutations of the ACTH-R have been described in patients with FGD in segregation with the disease. The functional characterization of these mutations has been prevented by difficulties in expressing human ACTH-R in cells that lack endogenous melanocortin receptor activity. To overcome these difficulties we used Y6 cells, a mutant variant of the Y1 cell line, which possesses a non-expressed ACTH-R gene allowing the functional study without any background activity. Our results demonstrated that the several mutations of the ACTH-R found in FGD result in an impaired cAMP response or loss of sensitivity to ACTH stimulation. An ACTH-binding study showed an impairment of ligand binding with loss of the high affinity site in most of the mutations studied.

High frequency of mutation G377S in Brazilian type 3 Gaucher disease patients

Gaucher disease (GD), the most prevalent lysosome storage disorder, presents an autosomal recessive mode of inheritance. It is a paradigm for therapeutic intervention in medical genetics due to the existence of effective enzyme replacement therapy. We report here the analysis of GD in 262 unrelated Brazilian patients, carried out in order to establish the frequency of the most common mutations and to provide prognostic information based on genotype-phenotype correlations. Among 247 type 1 GD patients, mutation N370S was detected in 47% of all the alleles, but N370S/N370S homozygosity was found in only 10% of the patients, a much lower frequency than expected, suggesting that most individuals presenting this genotype may not receive medical attention. Recombinant alleles were detected at a high frequency: 44% of the chromosomes bearing mutation L444P had other mutations derived from the pseudogene sequence, present in 25% of patients. Three neuronopathic type 2 patients were homozygous for L444P, all presenting additional mutations (E326K or recombinant alleles) that probably lead to the more severe phenotypes. Six children, classified as type 1 GD patients, had a L444P/L444P genotype, showing that neuronopathic symptoms may only manifest later in life. This would indicate the need for a higher treatment dose during enzyme replacement therapy. Finally, mutation G377S was present in 4 homozygous type 1 patients and also in compound heterozygosity in 5 (42%) type 3 patients. These findings indicate that G377S cannot be unambiguously classified as mild and suggest an allele-dose effect for this mutation.

Five years results after intrafamilial kidney post-transplant in a case of familial hypomagnesemia due to a claudin-19 mutation

Introduction: Familial Hypomagnesaemia with hypercalciuria and nephrocalcinosis, with severe ocular impairment secondary to claudin-19 mutation, is a rare recessive autossomic disorder. Its spectrum includes renal Mg2+ wasting, medullary nephrocalcinosis and progressive chronic renal failure in young people. Objective: To report a case of kidney transplantation father to daughter in a familial occurrence of severe bilateral nephrocalcinosis associated with ocular impairment in a non-consanguineous Brazilian family, in which two daughters had nephrocalcinosis and severe retinopathy. Methods: The index case, a 19 years-old female, had long-lasting past medical history of recurrent urinary tract infections, and the abdominal X-ray revealed bilateral multiple renal calcifications as well as ureteral lithiasis, and she was under haemodialysis. She had the diagnosis of retinitis pigmentosa in the early neonatal period. The other daughter (13 years-old) had also nephrocalcinosis with preserved kidney function, retinopathy with severe visual impairment, and in addition, she exhibited hypomagnesaemia = 0.5 mg/dL and hypercalciuria. The other family members (mother, father and son) had no clinical disease manifestation. Mutation analysis at claudin-19 revealed two heterozygous missense mutations (P28L and G20D) in both affected daughters. The other family members exhibited mutant monoallelic status. In despite of that, the index case underwent intrafamilial living donor kidney transplantation (father). Conclusion: In conclusion, the disease was characterized by an autosomal recessive compound heterozygous status and, after five years of donation the renal graft function remained stable without recurrence of metabolic disturbances or nephrocalcinosis. Besides, donor single kidney Mg2+ and Ca2+ homeostasis associated to monoallelic status did not affect the safety and the usual living donor post-transplant clinical course.

Recurrence of atrial septal defect in three generations

Beginning with a patient presenting with an atrial septal defect (ASD) of the secundum type, the genealogy was identified in four affected individuals who belonged to three successive generations of the same family. The defects were visually confirmed in all individuals and were found to be anatomically similar. No other congenital malformations were present in these individuals. The genealogy was identified in 1972, when ASD recurred in two generations, and it was concluded that the mechanism of transmission was autosomal recessive. The fifth individual, identified 21 years later, and having an anomaly identical to that of the others, was the child of a couple who had no consaguinity and whose mother was a member of the previously studied genealogy. Considering the absence of phenotype in the parents and the rarity of the ASD gene in the general population, the occurrence of the uniparental disomy for this family nucleus, and the same autosomal recessive mechanism of transmission by this affected individual is possible. This study reports the familial occurrence of ASD by genetic mechanisms of transmission, emphasizing the necessity for genetic-clinical studies in members of the familial nucleus in order to detect new carriers, who usually are asymptomatic, thereby allowing for early and adequate treatment of individuals who may be affected.

Síndrome de Peutz-Jeghers: relato de caso

Peutz-Jeghers syndrome (PJS) is a dominant autosomal inherited disorder characterized by intestinal hamartomatous polyps in association with mucocutaneous melanocytic maculae. This syndrome is rare, and the frequency reaches from 1 in 60,000 to 1 in 300,000 people in the USA. The symptom presentations vary greatly in this disease. Some patients require minor clinical treatment while others undergo many hospitalizations and surgical treatments. In addition, patients with PJS have an increased risk for developing a variety of malignant tumors. The aim of the present study was to report one case studied of Peutz-Jeghers syndrome.

Arthrogryposis in Murrah buffaloes in southern Brazil

Congenital arthrogryposis is described in a Murrah buffalo herd. The disease was characterized by curvature and multiple articular rigidity of the hindlimbs or of all limbs without associated defects except for one case of brachygnatia. Histologically there was reduction of motor neurons from the ventral horns of the spinal cord and hypoplasia of the limb muscles. Analysis of the herd breeding records suggests that the disease is genetically transmitted by an autosomal recessive trait.

A novel polymorphism in the coding region of the vasopressin type 2 receptor gene

Nephrogenic diabetes insipidus (NDI) is a rare disease characterized by renal inability to respond properly to arginine vasopressin due to mutations in the vasopressin type 2 receptor (V2(R)) gene in affected kindreds. In most kindreds thus far reported, the mode of inheritance follows an X chromosome-linked recessive pattern although autosomal-dominant and autosomal-recessive modes of inheritance have also been described. Studies demonstrating mutations in the V2(R) gene in affected kindreds that modify the receptor structure, resulting in a dys- or nonfunctional receptor have been described, but phenotypically indistinguishable NDI patients with a structurally normal V2(R) gene have also been reported. In the present study, we analyzed exon 3 of the V2(R) gene in 20 unrelated individuals by direct sequencing. A C®T alteration in the third position of codon 331 (AGC®AGT), which did not alter the encoded amino acid, was found in nine individuals, including two unrelated patients with NDI. Taken together, these observations emphasize the molecular heterogeneity of a phenotypically homogeneous syndrome

Structure and function of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs), and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs).