Clinical report and genetic analysis of rare premature infant nephronophthisis caused by biallelic TTC21B variants.

BACKGROUND: Nephronophthisis (NPHP) is a genetically heterogeneous disease that can lead to end-stage renal disease (ESRD) in children. The TTC21B variant is associated with NPHP12 and mainly characterized by cystic kidney disease, skeletal malformation, liver fibrosis, and retinopathy. Affected patients range from children to adults. Some patients experience ESRD in infancy or early childhood, but clinical reports on neonatal patients are rare. We report a case of NPHP12 in a premature infant and analyze its genetic etiology. METHODS: Trio-whole exome sequencing analysis was performed on the patient and her parents; bioinformatics software was used to predict and analyze the hazards of the variants. Sanger sequencing was performed to verify variants. We calculated the free energy between mutant IFT139 and the IFT121-IFT122-IFT43 complex structure using molecular dynamics (MD). Finally, the clinical and genetic characteristics of patients with hotspot variant Cys518Arg were reviewed. RESULTS: Genetic analysis revealed compound-heterozyous TTC21B variants in the patient, c.497delA (p.Lys166fs*36) and c.1552T>C (p.Cys518Arg). Her father and mother had heterozygous c.497delA (p.Lys166fs*36) and heterozygous c.1552T>C (p.Cys518Arg), respectively. Cys518Arg represents a hotspot variant, and the MD calculation results show that this can reduce the structural stability of the IFT121-IFT122-IFT139-IFT43 complex structure. A literature review showed that Cys518Arg might lead to the early occurrence of ESRD. CONCLUSIONS: Compound-heterozygous TTC21B variants underlie the phenotype in this patient. Thus, Cys518Arg may be a hotspot variant in the Chinese population. Genetic testing should be recommended for NPHP in neonates and early infants.

A single heterozygous nonsense mutation in the TTC21B gene causes adult-onset nephronophthisis 12: A case report and review of literature.

BACKGROUND: Nephronophthisis type 12 (NPHP 12) is a rare cilia-related cystic kidney disease, caused by TTC21B mutation, mainly involving the kidneys, which generally occurs in children. Our study aimed to illustrate its clinical, pathological and genetic characteristics by reporting an adult-onset case of NPHP 12 caused by a single heterozygous nonsense mutation of TTC21B confirmed by renal histology and whole exome sequencing and reviewing related literature with a comparative analysis of the clinical features of each case. It will further increase the recognition of this rare kidney genetic disease, which sometimes can manifest as an adult disease. RESULTS: A 33-years-old man showed a chronic disease course, and he exhibited slight renal dysfunction (CKD stage 3, eGFR = 49 ml/[min* 1.73 m2]) with renal tubular proteinuria, without any extrarenal manifestations, congenital malformation history of kidney disease, or family hereditary disease. Renal histological findings showed substantial interstitial fibrosis with some irregular and tortuous tubules with complex branches and segmental thickening and splitting of the tubular basement membrane. The patient was diagnosed with chronic interstitial nephritis for an unknown reason clinically. Further genetic analysis revealed a single heterozygous nonsense mutation in the TTC21B gene and NPHP 12 was diagnosed finally. CONCLUSION: A single heterozygous mutation in the TTC21B gene may cause atypical NPHP12, which had a relatively later onset and milder clinical symptoms without developmental abnormalities. Therefore, for unexplained adult-onset chronic interstitial nephritis with unusual changes of renal tubules and interstitial fibrosis, even without a clear history of hereditary kidney disease, genetic testing is still recommended. The correct diagnosis of this rare adult-onset hereditary nephropathy can avoid unnecessary treatment.

Biallelic mutations of TTC12 and TTC21B were identified in Chinese patients with multisystem ciliopathy syndromes.

BACKGROUND: Abnormalities in cilia ultrastructure and function lead to a range of human phenotypes termed ciliopathies. Many tetratricopeptide repeat domain (TTC) family members have been reported to play critical roles in cilium organization and function. RESULTS: Here, we describe five unrelated family trios with multisystem ciliopathy syndromes, including situs abnormality, complex congenital heart disease, nephronophthisis or neonatal cholestasis. Through whole-exome sequencing and Sanger sequencing confirmation, we identified compound heterozygous mutations of TTC12 and TTC21B in six affected individuals of Chinese origin. These nonsynonymous mutations affected highly conserved residues and were consistently predicted to be pathogenic. Furthermore, ex vivo cDNA amplification demonstrated that homozygous c.1464 + 2 T > C of TTC12 would cause a whole exon 16 skipping. Both mRNA and protein levels of TTC12 were significantly downregulated in the cells derived from the patient carrying TTC12 mutation c.1464 + 2 T > C by real-time qPCR and immunofluorescence assays when compared with two healthy controls. Transmission electron microscopy analysis further identified ultrastructural defects of the inner dynein arms in this patient. Finally, the effect of TTC12 deficiency on cardiac LR patterning was recapitulated by employing a morpholino-mediated knockdown of ttc12 in zebrafish. CONCLUSIONS: To the best of our knowledge, this is the first study reporting the association between TTC12 variants and ciliopathies in a Chinese population. In addition to nephronophthisis and laterality defects, our findings demonstrated that TTC21B should also be considered a candidate gene for biliary ciliopathy, such as TTC26, which further expands the phenotypic spectrum of TTC21B deficiency in humans.

Genetic Interaction of Thm2 and Thm1 Shapes Postnatal Craniofacial Bone.

Ciliopathies are genetic syndromes that link skeletal dysplasias to the dysfunction of primary cilia. Primary cilia are sensory organelles synthesized by intraflagellar transport (IFT)-A and B complexes, which traffic protein cargo along a microtubular core. We have reported that the deletion of the IFT-A gene, Thm2, together with a null allele of its paralog, Thm1, causes a small skeleton with a small mandible or micrognathia in juvenile mice. Using micro-computed tomography, here we quantify the craniofacial defects of Thm2-/-; Thm1aln/+ triple allele mutant mice. At postnatal day 14, triple allele mutant mice exhibited micrognathia, midface hypoplasia, and a decreased facial angle due to shortened upper jaw length, premaxilla, and nasal bones, reflecting altered development of facial anterior-posterior elements. Mutant mice also showed increased palatal width, while other aspects of the facial transverse, as well as vertical dimensions, remained intact. As such, other ciliopathy-related craniofacial defects, such as cleft lip and/or palate, hypo-/hypertelorism, broad nasal bridge, craniosynostosis, and facial asymmetry, were not observed. Calvarial-derived osteoblasts of triple allele mutant mice showed reduced bone formation in vitro that was ameliorated by Hedgehog agonist, SAG. Together, these data indicate that Thm2 and Thm1 genetically interact to regulate bone formation and sculpting of the postnatal face. The triple allele mutant mice present a novel model to study craniofacial bone development.

Biallelic variants in TTC21B as a rare cause of early-onset arterial hypertension and tubuloglomerular kidney disease.

Monogenic disorders of the kidney typically affect either the glomerular or tubulointerstitial compartment producing a distinct set of clinical phenotypes. Primary focal segmental glomerulosclerosis (FSGS), for instance, is characterized by glomerular scarring with proteinuria and hypertension while nephronophthisis (NPHP) is associated with interstitial fibrosis and tubular atrophy, salt wasting, and low- to normal blood pressure. For both diseases, an expanding number of non-overlapping genes with roles in glomerular filtration or primary cilium homeostasis, respectively, have been identified. TTC21B, encoding IFT139, however has been associated with disorders of both the glomerular and tubulointerstitial compartment, and linked with defective podocyte cytoskeleton and ciliary transport, respectively. Starting from a case report of extreme early-onset hypertension, proteinuria, and progressive kidney disease, as well as data from the Genomics England 100,000 Genomes Project, we illustrate here the difficulties in assigning this mixed phenotype to the correct genetic diagnosis. Careful literature review supports the notion that biallelic, often hypomorph, missense variants in TTC21B are commonly associated with early-onset hypertension and histological features of both FSGS and NPHP. Increased clinical recognition of this mixed glomerular and tubulointerstitial disease with often mild or absent features of a typical ciliopathy as well as inclusion of TTC21B on gene panels for early-onset arterial hypertension might shorten the diagnostic odyssey for patients affected by this rare tubuloglomerular kidney disease.

A case of proliferative glomerulosclerosis with compound heterozygous TTC21B mutations.

Mesangial proliferative glomerulonephritis (MsPGN) is the most common clinicopathologic feature of the primary glomerulonephritis. The hereditary susceptibility to MsPGN is rather complex. In this report, a Chinese case of proliferative glomerulosclerosis was recruited. Renal biopsy revealed extensive glomerulosclerosis with mesangial hypertrophy, and tubular atrophy and dilatation. Whole exome sequencing (WES) revealed compound heterozygous variants in TTC21B gene, which were confirmed by Sanger sequencing. The variants in TTC21B gene were the molecular pathogenic basis of this disorder, and this case help to understand the correlation of genotype and phenotypes of TTC21B mutations.

Case Report: Homozygous Pathogenic Variant P209L in the TTC21B Gene: A Rare Cause of End Stage Renal Disease and Biliary Cirrhosis Requiring Combined Liver-Kidney Transplantation. A Case Report and Literature Review.

Background: Ciliopathies are rare diseases causing renal and extrarenal manifestations. Here, we report the case of a ciliopathy induced by a homozygous pathogenic variant in the TTC21B gene. Case Description: A 47-year-old patient started hemodialysis for chronic kidney disease (CKD) of unknown origin. She presented with early onset of hypertension, pre-eclampsia, myopia and cirrhosis. Renal biopsy showed mild interstitial fibrosis, tubular atrophy, and moderate arteriosclerosis while liver pathology demonstrates grade B biliary cirrhosis. Family history revealed several cases of early-onset severe hypertension and one case of end-stage renal disease (ESRD) needing kidney transplantation at twenty years of age. Clinical exome sequencing showed homozygosis for the pathogenic variant c.626C>T (p.Pro209Leu) in the TTC21B gene. The patient underwent combined liver-renal transplantation with an excellent renal and hepatic graft outcome. Conclusions: TTC21B gene mutations can lead heterogeneous to clinical manifestations and represent an underappreciated cause of ESRD. The paradigm in diagnosis of CKD of early onset and/or of unknown origin is changing and genetic counseling should be performed in all patients and families that meet those criteria. Renal or combined liver-renal transplantation represents the best option for patients suffering from those diseases in terms of prognosis and quality of life.

Tubuloglomerular Disease With Cone-Shaped Epiphyses Associated With Hypomorphic Variant and a Novel p.Cys14Arg in the TTC21B Gene: A Case Report.

Monogenic nephrotic syndrome (NS) is associated with a resistance to initial glucocorticoid therapy and causative variants, which may be found in several genes influencing podocyte stability and kidney development. The TTC21B gene, which encodes the retrograde intraflagellar transport protein IFT139, is found mostly in association with ciliopathies in humans. The role of this protein in podocyte cytoskeleton stability was confirmed later and the mutated TTC21B also may be associated with proteinuric diseases, such as nephrotic syndrome. Our patient manifested as an infant with brachydactyly, nephrotic-range proteinuria, and renal tubular acidosis, and a kidney biopsy revealed focal segmental glomerulosclerosis (FSGS). Multiple phalangeal cone-shaped epiphyses of the hand were seen on X-ray. Next-generation sequencing revealed the well-described p.Pro209Leu heterozygous variant and a novel heterozygous p.Cys14Arg variant in the TTC21B gene. Our finding confirmed that the causative variants in the TTC21B gene may contribute to a spectrum of clinical features, such as glomerular proteinuric disease with tubulointerstitial involvement and skeletal abnormalities.

IFT-A deficiency in juvenile mice impairs biliary development and exacerbates ADPKD liver disease.

Polycystic liver disease (PLD) is characterized by the growth of numerous biliary cysts and presents in patients with autosomal dominant polycystic kidney disease (ADPKD), causing significant morbidity. Interestingly, deletion of intraflagellar transport-B (IFT-B) complex genes in adult mouse models of ADPKD attenuates the severity of PKD and PLD. Here we examine the role of deletion of an IFT-A gene, Thm1, in PLD of juvenile and adult Pkd2 conditional knockout mice. Perinatal deletion of Thm1 resulted in disorganized and expanded biliary regions, biliary fibrosis, increased serum bile acids, and a shortened primary cilium on cytokeratin 19+ (CK19+) epithelial cells. In contrast, perinatal deletion of Pkd2 caused PLD, with multiple CK19+ epithelial cell-lined cysts, fibrosis, lengthened primary cilia, and increased Notch and ERK signaling. Perinatal deletion of Thm1 in Pkd2 conditional knockout mice increased hepatomegaly, liver necrosis, as well as serum bilirubin and bile acid levels, indicating enhanced liver disease severity. In contrast to effects in the developing liver, deletion of Thm1 alone in adult mice did not cause a biliary phenotype. Combined deletion of Pkd2 and Thm1 caused variable hepatic cystogenesis at 4 months of age, but differences in hepatic cystogenesis between Pkd2- and Pkd2;Thm1 knockout mice were not observed by 6 months of age. Similar to juvenile PLD, Notch and ERK signaling were increased in adult Pkd2 conditional knockout cyst-lining epithelial cells. Taken together, Thm1 is required for biliary tract development, and proper biliary development restricts PLD severity. Unlike IFT-B genes, Thm1 does not markedly attenuate hepatic cystogenesis, suggesting differences in regulation of signaling and cystogenic processes in the liver by IFT-B and -A. Notably, increased Notch signaling in cyst-lining epithelial cells may indicate that aberrant activation of this pathway promotes hepatic cystogenesis, presenting as a novel potential therapeutic target. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Thm2 interacts with paralog, Thm1, and sensitizes to Hedgehog signaling in postnatal skeletogenesis.

Mutations in the intraflagellar transport-A (IFT-A) gene, THM1, have been identified in skeletal ciliopathies. Here, we report a genetic interaction between Thm1, and its paralog, Thm2, in postnatal skeletogenesis. THM2 localizes to primary cilia, but Thm2 deficiency does not affect ciliogenesis and Thm2-null mice survive into adulthood. However, by postnatal day 14, Thm2-/-; Thm1aln/+ mice exhibit small stature and small mandible. Radiography and microcomputed tomography reveal Thm2-/-; Thm1aln/+ tibia are less opaque and have reduced cortical and trabecular bone mineral density. In the mutant tibial growth plate, the proliferation zone is expanded and the hypertrophic zone is diminished, indicating impaired chondrocyte differentiation. Additionally, mutant growth plate chondrocytes show increased Hedgehog signaling. Yet deletion of one allele of Gli2, a major transcriptional activator of the Hedgehog pathway, exacerbated the Thm2-/-; Thm1aln/+ small phenotype, and further revealed that Thm2-/-; Gli2+/- mice have small stature. In Thm2-/-; Thm1aln/+ primary osteoblasts, a Hedgehog signaling defect was not detected, but bone nodule formation was markedly impaired. This indicates a signaling pathway is altered, and we propose that this pathway may potentially interact with Gli2. Together, our data reveal that loss of Thm2 with one allele of Thm1, Gli2, or both, present new IFT mouse models of osteochondrodysplasia. Our data also suggest Thm2 as a modifier of Hedgehog signaling in postnatal skeletal development.

A novel heterotaxy gene: Expansion of the phenotype of TTC21B-spectrum disease.

TTC21B encodes the protein IFT139, a critical component of the retrograde transport system within the primary cilium. Biallelic, pathogenic TTC21B variants are associated with classic ciliopathy syndromes, including nephronophthisis, Jeune asphyxiating thoracic dystrophy, and Joubert Syndrome, with ciliopathy-spectrum traits such as biliary dysgenesis, primary ciliary dyskinesia, and situs inversus, and also with focal segmental glomerulosclerosis. We report a 9-year-old male with focal segmental glomerulosclerosis requiring kidney transplant, primary ciliary dyskinesia, and biliary dysgenesis, found by research-based exome sequencing to have biallelic pathogenic TTC21B variants. A sibling with isolated heterotaxy was found to harbor the same variants. This case highlights the phenotypic spectrum and unpredictable manifestations of TTC21B-related disease, and also reports the first association between TTC21B and heterotaxy, nominating TTC21B as an important new heterotaxy gene.

A Compound Heterozygous Mutation in the Ciliary Gene TTC21B Causes Nephronophthisis Type 12.

Nephronophthisis (NPHP) is one of the renal ciliopathies and is also a cystic renal disorder with an autosomal recessive inheritance, which usually progresses to end-stage renal disease (ESRD). It affects children, adolescents, and young adults. In approximately 15% of cases, the features of a ciliopathy syndrome, which include liver fibrosis, skeletal anomalies, retinal abnormalities, and neurodevelopmental delay, will be present. We describe a case of a 2-year-old male child with ESRD on hemodialysis and a family record of a similar condition (his brother). The clinical features of this child are succinctly summarized. The genetic study was conducted using whole exome sequencing. TTC21B mutational variants were detected in our patient who exhibited nephrotic-range proteinuria, focal segmental glomerulosclerosis, and tubulointerstitial lesions that evolved to ESRD. Compound heterozygous mutations, c.626c > t (p.P209L) in exon 6 and c.450 g > a (p.W150Ter) in exon 5, were uncovered. These findings are in line with the description of autosomal recessive NPHP type 12. Both clinical and pathological diagnoses of NPHP are critical, bearing in mind ESRD as well as its related extrarenal defining features. Identification of the pathogenic variants in the TTC21B gene assisted in the successful proof of the clinical diagnosis NPHP12 as well as providing information for formal suitable prenatal counseling.

Medullary Cystic Kidney Disease and Focal Segmental Glomerulosclerosis Caused by a Compound Heterozygous Mutation in TTC21B.

Mutations in the TTC21B gene have been identified in patients with nephronophthisis and were recently found in some patients with focal segmental glomerulosclerosis. We herein report a Japanese boy with end-stage renal disease due to medullary polycystic kidney disease and primary focal segmental glomerulosclerosis. Next-generation sequencing detected a new compound heterozygous missense mutation in the TTC21B gene. His renal pathological findings and gene mutations have not been previously reported in patients with ciliopathy. For children with severe renal dysfunction, mutations in the TTC21B gene cause both ciliopathy characterized by bilateral polycystic kidney disease and primary focal segmental glomerulosclerosis.

Mutations in TTC21B cause different phenotypes in two childhood cases in China.

AIM: The TTC21B gene is now known as causative of nephronophthisis-related ciliopathies (NPHP-RC). We reported two Chinese paediatric cases with end-stage renal disease and other phenotypes caused by the TTC21B gene mutations. METHODS: The clinical features of Chinese paediatric cases with NPHP-RC were summarized. Mutation analysis of the TTC21B gene was performed using next-generation sequencing. RESULTS: The two cases both had nephrotic proteinuria, renal failure, hypertension and abnormal liver function (or hepatic fibrosis). One case also presented situs inversus and short phalanges. They developed end-stage renal disease (ESRD) at 1 year old and 8 years old, respectively, when renal pathology both showed focal segmental glomerular sclerosis (FSGS) with tubulointerstitial lesions including interstitial fibrosis and atrophic tubules. Three novel disease-causing TTC21B mutations were identified. One case carried homozygous mutation c.2211 + 3A > G, while the other case carried compound heterozygous mutations c.1552 T > C (p.C518R) and c.1456dupA (p.R486KfsX22). CONCLUSION: Mutations in TTC21B cause a range of ciliopathy phenotypes in humans. We identified 3 novel TTC21B mutations in two Chinese paediatric cases that both presented end-stage renal disease and other different features. This is the first TTC21B mutations ever reported in China.

Ttc21b Is Required in Bergmann Glia for Proper Granule Cell Radial Migration.

Proper cerebellar development is dependent on tightly regulated proliferation, migration, and differentiation events. Disruptions in any of these leads to a range of cerebellar phenotypes from ataxia to childhood tumors. Animal models have shown that proper regulation of sonic hedgehog (Shh) signaling is crucial for normal cerebellar architecture, and increased signaling leads to cerebellar tumor formation. Primary cilia are known to be required for the proper regulation of multiple developmental signaling pathways, including Shh. Tetratricopeptide Repeat Domain 21B (Ttc21b) is required for proper primary cilia form and function, and is primarily thought to restrict Shh signaling. Here we investigated a role for Ttc21b in cerebellar development. Surprisingly, Ttc21b ablation in Bergmann glia resulted in the accumulation of ectopic granule cells in the lower/posterior lobes of the cerebellum and a reduction in Shh signaling. Ttc21b ablation in just Purkinje cells resulted in a similar phenotype seen in fewer cells, but across the entire extent of the cerebellum. These results suggest that Ttc21b expression is required for Bergmann glia structure and signaling in the developing cerebellum, and in some contexts, augments rather than attenuates Shh signaling.

Contribution of the TTC21B gene to glomerular and cystic kidney diseases.

Background: The TTC21B gene was initially described as causative of nephronophthisis (NPHP). Recently, the homozygous TTC21B p.P209L mutation has been identified in families with focal segmental glomerulosclerosis (FSGS) and tubulointerstitial lesions. Heterozygous TTC21B variants have been proposed as genetic modifiers in ciliopathies. We aimed to study the causative and modifying role of the TTC21B gene in glomerular and cystic kidney diseases. Methods: Mutation analysis of the TTC21B gene was performed by massive parallel sequencing. We studied the causative role of the TTC21B gene in 17 patients with primary diagnosis of FSGS or NPHP and its modifying role in 184 patients with inherited glomerular or cystic kidney diseases. Results: Disease-causing TTC21B mutations were identified in three families presenting nephrotic proteinuria with FSGS and tubulointerstitial lesions in which some family members presented hypertension and myopia. Two families carried the homozygous p.P209L and the third was compound heterozygous for the p.P209L and a novel p.H426D mutation. Rare heterozygous TTC21B variants predicted to be pathogenic were found in five patients. These TTC21B variants were significantly more frequent in renal patients compared with controls (P = 0.0349). Two patients with a heterozygous deleterious TTC21B variant in addition to the disease-causing mutation presented a more severe phenotype than expected. Conclusions: Our results confirm the causal role of the homozygous p.P209L TTC21B mutation in two new families with FSGS and tubulointerstitial disease. We identified a novel TTC21B mutation demonstrating that p.P209L is not the unique causative mutation of this nephropathy. Thus, TTC21B mutation analysis should be considered for the genetic diagnosis of families with FSGS and tubulointerstitial lesions. Finally, we provide evidence that heterozygous deleterious TTC21B variants may act as genetic modifiers of the severity of glomerular and cystic kidney diseases.