Mutations in the Gene That Encodes the F-Actin Binding Protein Anillin Cause FSGS

FSGS is characterized by segmental scarring of the glomerulus and is a leading cause of kidney failure. Identification of genes causing FSGS has improved our understanding of disease mechanisms and points to defects in the glomerular epithelial cell, the podocyte, as a major factor in disease pathogenesis. Using a combination of genome-wide linkage studies and whole-exome sequencing in a kindred with familial FSGS, we identified a missense mutation R431C in anillin (ANLN), an F-actin binding cell cycle gene, as a cause of FSGS. We screened 250 additional families with FSGS and found another variant, G618C, that segregates with disease in a second family with FSGS. We demonstrate upregulation of anillin in podocytes in kidney biopsy specimens from individuals with FSGS and kidney samples from a murine model of HIV-1–associated nephropathy. Overexpression of R431C mutant ANLN in immortalized human podocytes results in enhanced podocyte motility. The mutant anillin displays reduced binding to the slit diaphragm–associated scaffold protein CD2AP. Knockdown of the ANLN gene in zebrafish morphants caused a loss of glomerular filtration barrier integrity, podocyte foot process effacement, and an edematous phenotype. Collectively, these findings suggest that anillin is important in maintaining the integrity of the podocyte actin cytoskeleton.FSGS is a clinicopathologic entity that is characterized by nephrotic syndrome (NS), focally sclerotic glomeruli, and effacement of podocyte foot processes and is a common cause of ESRD.¹ The pathogenesis of FSGS has not been completely elucidated; however, recent advances in molecular genetics have provided evidence that disruption of podocyte structure and function is central to its pathogenesis. The highly specialized apparatus of the glomerular filtration barrier (GFB) is composed of the podocyte and slit diaphragm, the glomerular basement membrane, and specialized fenestrated endothelium. The podocyte and slit diaphragm play a central role in maintaining the structural and functional integrity of the GFB. This is evidenced by the number of genetic mutations found in familial FSGS and NS; many of these gene products contribute to signaling at the podocyte slit diaphragm or localize to the podocyte cytoskeleton.^2–8 However, the entire repertoire of genes and proteins that are important in maintaining the functional integrity of the GFB remains unknown. It is estimated that >90% of the genetic causes of hereditary FSGS and NS remain unknown⁹; thus, the study of large kindreds remains an invaluable tool for unraveling the complexity of the molecular interactions that are responsible for maintaining the integrity of the podocyte and GFB.We identified a pedigree from the United States with autosomal dominant (AD) FSGS. Linkage analysis was carried out and we obtained suggestive multipoint logarithm of odds (LOD) scores of 1.7, 1.7, and 1.8 on chromosomes 2p, 5p, and 7p respectively. Whole-exome sequencing identified a deleterious heterozygous mutation R431C in ANLN, the gene encoding the F-actin binding protein anillin. We found another variant G618C that segregates with disease in a second family with FSGS. Anillin is important in cytokinesis and it interacts with key proteins such as CD2AP and mDia2 during cell division.^10–13 In addition, it regulates cell growth by its interaction with the promigratory and prosurvival phosphoinositide 3 kinase/AKT (protein kinase B) pathway.^10–13 Overexpression of the mutant anillin in immortalized human podocytes causes increased podocyte motility compared with wild-type overexpression. In contrast with intact anillin, the mutant displays a significantly reduced binding affinity to CD2AP. These findings reemphasize the importance of podocyte cell integrity, cell survival pathways, and cell migration in the pathogenesis of FSGS.     

A novel mutation in LMX1B gene causes nail-patella syndrome in a large Chinese family

We conducted clinical and genetic studies in a large Chinese family with nail-patella syndrome (NPS) involving multi-organ (such as limb, renal and eye) and investigated the functional consequences of a novel LMX1B mutation identified in the family. Twenty individuals at risk for inheriting NPS in the Chinese family participated in the study and a physical examination was performed and blood was drawn for DNA extraction. Linkage analysis and mutation screening of LMX1B gene were performed and the functional study in vitro for the mutation was conducted by luciferase assay. The disease phenotype of this family was linked to D9S290 with LOD Score=5.8 at theta=0; a novel mutation 742 A>G (R248G) within the homeodomain was found in a conserved site and co-segregated with the disease phenotype of the family. The functional study in vitro by luciferase assay indicated that the R248G mutation within the binding domain of the gene affected the transactivation. This is the first report that a mutation in the LMX1B gene causes NPS in a Chinese population, which will expand the spectrum of mutations in the LMX1B gene and provide insight into the underlining pathology of NPS.     

A Rare Autosomal Dominant Variant in Regulator of Calcineurin Type 1 (RCAN1) Gene Confers Enhanced Calcineurin Activity and May Cause FSGS

Open in a separate window     

Mutations in Profilin 1 Cause Early-Onset Paget's Disease of Bone With Giant Cell Tumors

Paget's disease of bone (PDB) is a late-onset chronic progressive bone disease characterized by abnormal activation of osteoclasts that results in bone pain, deformities, and fractures. PDB is very rare in Asia. A subset of PDB patients have early onset and can develop malignant giant cell tumors (GCTs) of the bone (PDB/GCTs), which arise within Paget bone lesions; the result is a significantly higher mortality rate. SQSTM1, TNFRSF11A, OPG, VCP, and HNRNPA2B1 have been identified as pathogenic genes of PDB, and ZNF687 is the only confirmed gene to date known to cause PDB/GCT. However, the molecular mechanism underlying PDB/GCT has not been fully elucidated. Here, we investigate an extended Chinese pedigree with eight individuals affected by early-onset and polyostotic PDB, two of whom developed GCTs. We identified a heterozygous 4-bp deletion in the Profilin 1 (PFN1) gene (c.318_321delTGAC) by genetic linkage analysis and exome sequencing for the family. Sanger sequencing revealed another heterozygous 1-bp deletion in PFN1 (c.324_324delG) in a sporadic early-onset PDB/GCT patient, further proving its causative role. Interestingly, a heterozygous missense mutation of PFN1 (c.335 T > C) was identified in another PDB/GCT family, revealing that not only deletion but also missense mutations in PFN1 can cause PDB/GCT. Furthermore, we established a Pfn1-mutated mouse model (C57BL/6J mice) and successfully obtained Pagetic phenotypes in heterozygous mice, verifying loss of function of PFN1 as the cause of PDB/GCT development. In conclusion, our findings reveal mutations in PFN1 as the pathological mechanism in PDB/GCT, and we successfully established Pfn1-mutated mice as a suitable animal model for studying PDB-associated pathological mechanisms. The identification of PFN1 mutations has great diagnostic value for identifying PDB individuals predisposed toward developing GCTs. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Autosomal-Recessive Mutations in SLC34A1 Encoding Sodium-Phosphate Cotransporter 2A Cause Idiopathic Infantile Hypercalcemia

Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis. Recently, mutations in the vitamin D catabolizing enzyme 25-hydroxyvitamin D₃-24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of the active metabolite 1,25-(OH)₂D₃. In a subgroup of patients who presented in early infancy with renal phosphate wasting and symptomatic hypercalcemia, mutations in CYP24A1 were excluded. Four patients from families with parental consanguinity were subjected to homozygosity mapping that identified a second IIH gene locus on chromosome 5q35 with a maximum logarithm of odds (LOD) score of 6.79. The sequence analysis of the most promising candidate gene, SLC34A1 encoding renal sodium-phosphate cotransporter 2A (NaPi-IIa), revealed autosomal-recessive mutations in the four index cases and in 12 patients with sporadic IIH. Functional studies of mutant NaPi-IIa in Xenopus oocytes and opossum kidney (OK) cells demonstrated disturbed trafficking to the plasma membrane and loss of phosphate transport activity. Analysis of calcium and phosphate metabolism in Slc34a1-knockout mice highlighted the effect of phosphate depletion and fibroblast growth factor-23 suppression on the development of the IIH phenotype. The human and mice data together demonstrate that primary renal phosphate wasting caused by defective NaPi-IIa function induces inappropriate production of 1,25-(OH)₂D₃ with subsequent symptomatic hypercalcemia. Clinical and laboratory findings persist despite cessation of vitamin D prophylaxis but rapidly respond to phosphate supplementation. Therefore, early differentiation between SLC34A1 (NaPi-IIa) and CYP24A1 (24-hydroxylase) defects appears critical for targeted therapy in patients with IIH.     

Fixation of Vancouver B1 peri-prosthetic fractures by broad metal plates without the application of strut allografts

The use of allograft struts and cerclage wire, possibly augmented by plate fixation, for the treatment of Vancouver type-B1 peri-prosthetic fractures around a total hip replacement has been strongly advocated. We examined our results using plate fixation without allograft struts and compared them with the results of the use of struts alone or when combined with plate fixation. Of 20 consecutive patients with type-B1 fractures treated by open reduction and plate fixation, 19 were available for follow-up. The fractures healed in 18 patients with a mean time to weight-bearing of ten weeks (4 to 19). There were no cases of infection or malunion. Nonunion occurred in one patient and required a second plate fixation to achieve union. Safe, cost-effective treatment of Vancouver type-B1 fractures can be performed by plate fixation without the addition of cortical struts. This procedure may allow earlier weight-bearing than allograft strut fixation alone.     

囊泡型H+-ATPase B1 亚基的突变对大鼠内髓集合管细胞H+-ATPase结构和泵氢功能的影响

目的 研究致遗传性远端肾小管酸中毒（dRTA）的囊泡型H+-ATPase B1 亚基（ATP6V1B1）的点突变对大鼠内髓集合管（IMCD）细胞H+-ATPase结构和泵氢功能的影响。 方法 模拟致人类遗传性dRTA的 B1亚基点突变构建野生型（WT）和7种突变型（M)质粒,转染大鼠IMCD细胞并筛选稳定表达绿色荧光蛋白（GFP）-B1 M和GFP-B1 WT的IMCD细胞系。应用免疫荧光、免疫蛋白印迹法、ATP-NADH 耦合实验和快速酸负荷后不依赖钠的细胞内pH的变化,来观察GFP-B1 M和GFP-B1 WT在细胞内的分布,及其与H+-ATPase其他（E、H和c）亚基结合能力对ATP酶活性和H+-ATPase 泵氢功能的影响。 结果 GFP-B1 WT在转染细胞中呈囊泡样分布,与H+-ATPase的分布一致;而GFP-B1 M 则为弥散分布。免疫沉淀结果显示只有GFP-B1 WT融合蛋白能和其他的H+-ATPase 亚基（E、H和c）结合形成复合物,而 GFP-B1 M融合蛋白无此作用。ATP酶活性只有在GFP-B1 WT转染细胞株的免疫沉淀产物中存在,在GFP-B1 M转染细胞株的免疫沉淀产物中不存在。在GFP-B1 M 转染的IMCD细胞快速酸负荷后H+-ATPase介导的钠不依赖pHi 的恢复受到显著抑制[pHi的恢复率（pH U/min）在L81P、R124W、M174R、P275R、G316E、P346R、G364S GFP-B1M转染的IMCD细胞分别为0.007±0.002、0.004±0.002、0.002±0.002、0.003±0.002、0.006±0.004、0.009±0.004、0.015±0.006,P < 0.05,n = 5]。而GFP-B1 WT转染的IMCD细胞pHi的恢复率与未转染IMCD细胞相似[（0.040±0.006） pH U/min],且能被1 μmol/L巴弗洛霉素（H+-ATPase特异性抑制剂）所抑制。 结论 遗传性dRTA囊泡型H+-ATPase B1 亚基点突变影响GFP-B1融合蛋白与其他亚基正常结合组装形成完整的H+-ATPase,并抑制H+-ATPase 的泌酸功能。     

HSD11B1 polymorphisms predicted bone mineral density and fracture risk in postmenopausal women without a clinically apparent hypercortisolemia

IntroductionEndogenous glucocorticoid (GC) may participate in bone physiology, even in subjects with no glucocorticoid excess. 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) is a primary regulator catalyzing the reduction of inactive cortisone to active cortisol. To elucidate genetic relevance of HSD11B1 variants to vertebral fracture and osteoporosis, we investigated the potential involvement of six HSD11B1 SNPs in postmenopausal women.MethodsAll exons, their boundaries and the promoter region (approximately 1.5 kb) were directly sequenced in 24 individuals. Six polymorphisms were selected and genotyped in all study participants (n = 1329). BMD was measured using dual-energy X-ray absorptiometry.ResultsHSD11B1 + 16374C>T and + 27447G>C were associated with reduced vertebral fracture risk (p = 0.016 and 0.032, respectively). Two of these (LD block2) in intron 5 (rs1000283 and rs932335) were significantly associated with bone mineral density (BMD) at the femoral neck (p = 0.00005 and 0.0002, respectively). Specifically, HSD11B1 + 16374C>T and + 27447G>C polymorphisms were associated with higher BMD values of the femoral neck in multiple comparison (p = 0.0002 and 0.0004, respectively) and Bonferroni corrected significance level (97% power). Consistent with these results, HSD11B1-ht21 and -ht22 comprising both SNPs also showed the evidence of association with BMD values of the femoral neck (p_domiant = 0.0002 and p_recessive = 0.00005, respectively).ConclusionOur results provide preliminary evidence supporting an association of HSD11B1 with osteoporosis in postmenopausal women. Also, these findings demonstrate that + 16374C>T polymorphism may be useful genetic markers for bone metabolism.     

The Actions of Sevoflurane and Desflurane on the γ-Aminobutyric Acid Receptor Type A: Effects of TM2 Mutations in the α and β Subunits

Background: Previous studies have shown that specific amino acid residues in the putative second transmembrane segment (TM2) of the [gamma]-aminobutyric acid receptor type A (GABAA) receptor play a critical role in the enhancement of GABAA receptor function by halothane, enflurane, and isoflurane. However, very little is known about the actions of sevoflurane and desflurane on recombinant GABAA receptors. The aim of this study was to examine the effects of sevoflurane and desflurane on potentiation of GABA-induced responses in the wild-type GABAA receptor and in receptors mutated in TM2 of the [alpha]1, [alpha]2, or [beta]2 subunits.

Methods: GABAA receptor [alpha]1 or [alpha]2, [beta]2 or [beta]3, and [gamma]2s subunit cDNAs were expressed for pharmacologic study by transfection of human embryonic kidney 293 cells and assayed using the whole cell voltage clamp technique. Concentration-response curves and EC50 values for agonist were determined in the wild-type [alpha]1[beta]2[gamma]2s and [alpha]2[beta]3[gamma]2s receptors, and in receptors harboring mutations in TM2, such as [alpha]1(S270W)[beta]2[gamma]2s, [alpha]1[beta]2(N265W)[gamma]2s, and [alpha]2(S270I)[beta]3[gamma]2s. The actions of clinically relevant concentration of volatile anesthetics (isoflurane, sevoflurane, and desflurane) on GABA activated Cl- currents were compared in the wild-type and mutant GABAA receptors.

Results: Both sevoflurane and desflurane potentiated submaximal GABA currents in the wild-type GABAA [alpha]1[beta]2[gamma]2s receptor and [alpha]2[beta]3[gamma]2s receptor. Substitution of Ser270 in TM2 of the [alpha] subunit by a larger amino acid, tryptophan (W) or isoleucine (I), as in [alpha]1(S270W)[beta]2[gamma]2s and [alpha]2(S270I)[beta]3[gamma]2s, completely abolished the potentiation of GABA-induced currents by these anesthetic agents. In contrast, mutation of Asn265 in TM2 of the [beta] subunit to tryptophan (W) did not prevent potentiation of GABA-induced responses. The actions of sevoflurane and desflurane in the wild-type receptor and in mutated receptors were qualitatively and quantitatively similar to those observed for isoflurane.     

Evidence for the Involvement of Spinal Cord α1 Adrenoceptors in Nitrous Oxide-induced Antinociceptive Effects in Fischer Rats

Background: In a previous study, the authors found that nitrous oxide (N2O) exposure induces c-Fos (an immunohistochemical marker of neuronal activation) in spinal cord [gamma]-aminobutyric acid-mediated (GABAergic) neurons in Fischer rats. In this study, the authors sought evidence for the involvement of [alpha]1 adrenoceptors in the antinociceptive effect of N2O and in activation of GABAergic neurons in the spinal cord.

Methods: Adult male Fischer rats were injected intraperitoneally with [alpha]1 adrenoceptor antagonist, [alpha]2 adrenoceptor antagonist, opioid receptor antagonist, or serotonin receptor antagonist and, 15 min later, were exposed to either air (control) or 75% N2O. In some animals, nociception was investigated with the plantar test after 30 min of exposure, while in other animals, gas exposure was continued for 90 min and the spinal cord was examined for c-Fos immunostaining. In a separate experiment, animals were exposed to the above gases alone, after which the spinal cords were examined immunohistochemically for c-Fos and [alpha]1 adrenoceptor by double-staining methods.

Results: The antinociceptive effect of N2O was attenuated by prazosin (an [alpha]1 adrenoceptor antagonist), yohimbine (an [alpha]2 adrenoceptor antagonist), and naloxone (an opioid receptor antagonist) but not by methysergide and tropisetron (serotonin receptor antagonists). N2O exposure induced c-Fos expression in the spinal cord, which was blocked by prazosin and naloxone but not by other drugs. N2O-induced c-Fos expression was colocalized with [alpha]1 adrenoceptor immunoreactivity in laminae III-IV.     

Preferential Maternal Transmission of STX16-GNAS Mutations Responsible for Autosomal Dominant Pseudohypoparathyroidism Type Ib (PHP1B): Another Example of Transmission Ratio Distortion

Preferential transmission of a genetic mutation to the next generation, referred to as transmission ratio distortion (TRD), is well established for several dominant disorders, but underlying mechanisms remain undefined. Recently, TRD was reported for patients affected by pseudohypoparathyroidism type Ia or pseudopseudohypoparathyroidism. To determine whether TRD is observed also for autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP1B), we analyzed kindreds with the frequent 3-kb STX16 deletion or other STX16/GNAS mutations. If inherited from a female, these genetic defects lead to loss-of-methylation at exon A/B alone or at all three differentially methylated regions (DMR), resulting in parathyroid hormone (PTH)-resistant hypocalcemia and hyperphosphatemia and possibly resistance to other hormones. In total, we investigated 212 children born to 80 females who are unaffected carriers of a STX16/GNAS mutation (n = 47) or affected by PHP1B (n = 33). Of these offspring, 134 (63.2%) had inherited the genetic defect (p = .00012). TRD was indistinguishable for mothers with a STX16/GNAS mutation on their paternal (unaffected carriers) or maternal allele (affected). The mechanisms favoring transmission of the mutant allele remain undefined but are likely to include abnormalities in oocyte maturation. Search for mutations in available descendants of males revealed marginally significant evidence for TRD (p = .038), but these analyses are less reliable because many more offspring of males than females with a STX16/GNAS mutation were lost to follow-up (31 of 98 versus 6 of 218). This difference in follow-up is probably related to the fact that inheritance of a mutation from a male does not have clinical implications, whereas inheritance from an affected or unaffected female results in PHP1B. Lastly, affected PHP1B females had fewer descendants than unaffected carriers, but it remains unclear whether abnormal oocyte development or impaired actions of reproductive hormones are responsible. Our findings highlight previously not recognized aspects of AD-PHP1B that are likely to have implications for genetic testing and counseling. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Sequestosome 1 Mutations in Paget's Disease of Bone in Australia: Prevalence,Genotype/Phenotype Correlation,and a Novel Non‐UBA Domain Mutation (P364S) Associated With Increased NF‐κB Signaling Without Loss of Ubiquitin Binding

Previously reported Sequestosome 1(SQSTM1)/p62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin‐associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype/phenotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co‐transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF‐κB luciferase reporter gene. GST‐SQSTM1 (wildtype and mutant) proteins were used in pull‐down assays to compare monoubiquitin‐binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF‐κB activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF‐κB signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations.