Methylmalonic aciduria and homocystinuria, CblC type (OMIM #277400) is the most common disorder of cobalamin intracellular metabolism, an autosomal recessive disease, whose biochemical hallmarks are ...hyperhomocysteinemia, methylmalonic aciduria and low plasma methionine. Despite being a well-recognized disease for pediatricians, there is scarce awareness of its adult presentation. A thorough analysis and discussion of cobalamin C defect presentation in adult patients has never been extensively performed. This article reviews the published data and adds a new case of the latest onset of symptoms ever described for the disease.
We present the emblematic case of a 45-year-old male, describing the diagnostic odyssey he ventured through to get to the appropriate treatment and molecular diagnosis. Furthermore, available clinical, biochemical and molecular data from 22 reports on cases and case series were collected, resulting in 45 adult-onset CblC cases, including our own. We describe the onset of the disease in adulthood, encompassing neurological, psychiatric, renal, ophthalmic and thromboembolic symptoms. In all cases treatment with intramuscular hydroxycobalamin was effective in reversing symptoms. From a molecular point of view adult patients are usually compound heterozygous carriers of a truncating and a non-truncating variant in the MMACHC gene.
Adult onset CblC disease is a rare disorder whose diagnosis can be delayed due to poor awareness regarding its presenting insidious symptoms and biochemical hallmarks. To avoid misdiagnosis, we suggest that adult onset CblC deficiency is acknowledged as a separate entity from pediatric late onset cases, and that the disease is considered in the differential diagnosis in adult patients with atypical hemolytic uremic syndromes and/or slow unexplained decline in renal function and/or idiopathic neuropathies, spinal cord degenerations, ataxias and/or recurrent thrombosis and/or visual field defects, maculopathy and optic disc atrophy. Plasma homocysteine measurement should be the first line for differential diagnosis when the disease is suspected. To further aid diagnosis, it is important that genes belonging to the intracellular cobalamin pathway are included within gene panels routinely tested for atypical hemolytic uremic syndrome and chronic kidney disorders.
NOTCH1 mutations in chronic lymphocytic leukemia (CLL) lead to accumulation of NOTCH1 intracellular domain (NICD) and prolong signaling. These mutations associate with a more aggressive disease ...compared to wild-type (WT) CLL. In this work we demonstrate a bidirectional functional relationship between NOTCH1 and the B cell receptor (BCR) pathways. By using highly homogeneous cohorts of primary CLL cells, activation of NOTCH1 is shown to increase expression of surface IgM, as well as LYN, BTK, and BLNK, ultimately enhancing BCR signaling responses, including global mRNA translation. Upon BCR cross-linking, NOTCH1 itself is actively translated and increased on cell surface. Furthermore, BCR ligation induces calcium mobilization that can facilitate ligand-independent NOTCH1 activation. These data suggest that the two pathways are functionally linked, providing a rationale for dual inhibition strategies. Consistently, addition of the γ-secretase inhibitor DAPT to ibrutinib significantly potentiates its effects, both in vitro and in a short-term patient-derived xenograft model. While this observation may find limited applications in the CLL field, it is more relevant for Richter's Syndrome (RS) management, where very few successful therapeutic options exist. Treatment of RS-patient-derived xenografts (RS-PDX) with the combination of ibrutinib and DAPT decreases disease burden and increases overall survival.
Inherited kidney diseases are among the leading causes of kidney failure in children, resulting in increased mortality, high healthcare costs and need for organ transplantation. Next-generation ...sequencing technologies can help in the diagnosis of rare monogenic conditions, allowing for optimized medical management and therapeutic choices.
Clinical exome sequencing (CES) was performed on a cohort of 191 pediatric patients from a single institution, followed by Sanger sequencing to confirm identified variants and for family segregation studies.
All patients had a clinical diagnosis of kidney disease: the main disease categories were glomerular diseases (32.5%), ciliopathies (20.4%), CAKUT (17.8%), nephrolithiasis (11.5%) and tubular disease (10.5%). 7.3% of patients presented with other conditions. A conclusive genetic test, based on CES and Sanger validation, was obtained in 37.1% of patients. The highest detection rate was obtained for ciliopathies (74.4%), followed by nephrolithiasis (45.5%), tubular diseases (45%), while most glomerular diseases and CAKUT remained undiagnosed.
Results indicate that genetic testing consistently used in the diagnostic workflow of children with chronic kidney disease can (i) confirm clinical diagnosis, (ii) provide early diagnosis in the case of inherited conditions, (iii) find the genetic cause of previously unrecognized diseases and (iv) tailor transplantation programs.
In 2018, our center started a program to offer genetic diagnosis to patients with kidney and liver monogenic rare conditions, potentially eligible for organ transplantation. We exploited a clinical ...exome sequencing approach, followed by analyses of in silico gene panels tailored to clinical suspicions, obtaining detection rates in line with what reported in literature. However, a percentage of patients remains without a definitive genetic diagnosis. This work aims to evaluate the utility of NGS data re-analysis for those patients with an inconclusive or negative genetic test at the time of first analysis considering that (i) the advance of alignment and variant calling processes progressively improve the detection rate, limiting false positives and false negatives; (ii) gene panels are periodically updated and (iii) variant annotation may change over time.
114 patients, recruited between 2018 and 2020, with an inconclusive or negative NGS report at the time of first analysis, were included in the study. Re-alignment and variant calling of previously generated sequencing raw data were performed using the GenomSys Variant Analyzer software.
21 previously not reported potentially causative variants were identified in 20 patients. In most cases (n = 19), causal variants were retrieved out of the re-classification from likely benign to variants of unknown significance (VUS). In one case, the variant was included because of inclusion in the analysis of a newly disease-associated gene, not present in the original gene panel, and in another one due to the improved data alignment process. Whenever possible, variants were validated with Sanger sequencing and family segregation studies. As of now, 16 out of 20 patients have been analyzed and variants confirmed in 8 patients. Specifically, in two pediatric patients, causative variants were de novo mutations while in the others, the variant was present also in other affected relatives. In the remaining patients, variants were present also in non-affected parents, raising questions on their re-classification.
Overall, these data indicate that periodic and systematic re-analysis of negative or inconclusive NGS data reports can lead to new variant identification or reclassification in a small but significant proportion of cases, with benefits for patients' management.
The calcium-sensing receptor (CASR) gene encodes a G protein-coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants ...lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant c.2897_2898insCTGA, p.(Gln967*) (Q967*) identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants.
To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs.
Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT.
This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction.
A novel COLEC10 mutation in a child with 3MC syndrome Migliorero, Martina; Kalantari, Silvia; Bracciamà, Valeria ...
European journal of medical genetics,
December 2021, 2021-Dec, 2021-12-00, 20211201, Letnik:
64, Številka:
12
Journal Article
Recenzirano
3MC syndrome is an autosomal recessive disorder encompassing four rare disorders previously known as the Malpuech, Michels, Mingarelli and Carnevale syndromes. They are characterized by a variable ...spectrum of abnormalities, including facial dysmorphisms, along with genital, limb and vesico-renal anomalies. The syndrome was originally attributed to mutations in MASP1 and COLEC11, which code for proteins involved in the lectin complement pathway. More recently, mutations in COLEC10, a third gene coding for collectin CL-L1, were identified in a limited number of patients with 3MC syndrome. Here we describe a 4-years-old patient with typical 3MC phenotypic characteristics, including blepharophimosis, telecanthus, high arched eyebrows, fifth finger clinodactyly, sacral dimple and horseshoe kidney. Initial genetic analysis was based on clinical exome sequencing, where only MASP1 and COLEC11 genes are present, without evidence of pathogenic variants. Sanger sequencing of COLEC10 identified the homozygous frameshift variant c.807_810delCTGT; p.Cys270Serfs*33, which results in the loss of the natural stop codon. The resulting protein is 24 amino acids longer and lacks a conserved cysteine residue (Cys270), which could affect protein folding. Segregation studies confirmed that both parents were carriers for the variant: interestingly they originate from the same area of Apulia in southern Italy. Plasma levels of CL-L1 in the patient and her parents were within normal range, suggesting that this variant does not modify transcription or secretion. However, the variant affects the chemo-attractive feature of CL-L1, as HeLa cells migrate significantly less in response to the mutant protein compared to the wild-type one.
Background
A considerable minority of patients on waiting lists for kidney transplantation either have no diagnosis (and fall into the subset of
undiagnosed
cases) because kidney biopsy was not ...performed or histological findings were non-specific, or do not fall into any well-defined clinical category. Some of these patients might be affected by a previously unrecognised monogenic disease.
Methods
Through a multidisciplinary cooperative effort, we built an analytical pipeline to identify patients with chronic kidney disease (CKD) with a clinical suspicion of a monogenic condition or without a well-defined diagnosis. Following the stringent phenotypical and clinical characterization required by the flowchart, candidates meeting these criteria were further investigated by clinical exome sequencing followed by in silico analysis of 225 kidney-disease-related genes.
Results
By using an ad hoc web-based platform, we enrolled 160 patients from 13 different Nephrology and Genetics Units located across the Piedmont region over 15 months. A preliminary “remote” evaluation based on well-defined inclusion criteria allowed us to define eligibility for NGS analysis. Among the 138 recruited patients, 52 (37.7%) were children and 86 (62.3%) were adults. Up to 48% of them had a positive family history for kidney disease. Overall, applying this workflow led to the identification of genetic variants potentially explaining the phenotype in 78 (56.5%) cases.
Conclusions
These results underline the importance of clinical exome sequencing as a versatile and highly useful, non-invasive tool for genetic diagnosis of kidney diseases. Identifying patients who can benefit from targeted therapies, and improving the management of organ transplantation are further expected applications.
) is a member of the homeodomain-containing family of transcription factors located on 17q12.
deficiency is associated with a clinical syndrome (kidney and urogenital malformations, maturity-onset ...diabetes of the young, exocrine pancreatic insufficiency) and to an underdiagnosed liver involvement. Differently from
, the correlation between hepatocellular carcinoma (HCC) and germline
deficiency has been poorly evaluated.
Here, we report a novel case of a syndromic
-deficient paediatric patient that developed HCC with unique histopathological features characterised by neoplastic syncytial giant cells, which was observed only in one additional case of paediatric cholestatic liver disease of unknown origin.
Our case highlights the influence of
deficiency in liver disease progression and its putative association with a rare yet specific HCC histotype. We hypothesised that HCC could be secondary to the repressive effect of
variant on the
transcriptional activity.
Abstract
Background and Aims
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common single-gene disorder and the most frequent progressive kidney disease, which ultimately leads to ...kidney failure and renal replacement therapy. Almost 80% of cases of ADPKD are attributed to germline mutations in PKD1, even though at least one second somatic event such as the inactivation of the remaining wild-type PKD1 allele is required for the disease's manifestation. A significant portion of all variants identified in PKD1 is classified as “variant of unknown significance” (VUS) and understanding their functional impact may be of diagnostic importance for patients and their families. So, a proper experimental model to study the functional impact of different genetic lesions is needed to readily confirm their pathogenicity.
Method
The HEK293T cell line was modified to express an inducible Cas9 and afterwards transfected with a sgRNA targeting exon 15 of PKD1 to generate clones carrying homozygous or heterozygous nonsense variants, validated by Sanger sequencing. Transcript level and protein expression were evaluated, and then functional read-outs were set-up to validate the model. Heterozygous clones were used to introduce a second PKD1 variant, c.11614G>A p.(Glu3872Lys) in exon 42, using a modified CRISPR system called base editors, able to operate single-base substitutions (Figure 1A).
Results
Heterozygous (+/−) and homozygous (−/−) exon 15 PKD1 clones were generated. These cells showed a slight decrement in PKD1 mRNA levels, and PC1 protein loss was confirmed in full knock-out clones. Functionally, immunofluorescence staining highlighted a different cytoskeletal rearrangement in cells lacking PC1, which did not present actin protrusions at variance with WT and heterozygous cells. In agreement with these results, PKD1−/− clones showed reduced migration as demonstrated by wound healing assay. In starving conditions, PKD1−/− cells were characterized by increased viability, resistance to cell death and a disrupted autophagic pathway, as demonstrated by LC3BI-II conversion, which in PKD1−/− cells is diminished. Validation of the model is also supported by RNA sequencing data indicating that double knock-out cell lines display an upregulation of genes involved in proliferative pathways and epithelial-mesenchymal transition, as well as a down-regulation of genes involved in cytoskeletal organization.
We then used the heterozygous exon 15 clones to introduce a specific variant in exon 42, classified as C4 according to ACMG criteria. To do so, PKD1+/+ and PKD1+/− cell lines were transfected with a sgRNA guide that correctly positioned the Cytosine Base Editor. Using this approach we could generate a double knock-out cell line on exon 42 and a “double hit” cell line carrying a heterozygous variant on exon 15 and an homozygous one on exon 42, which represents a more likely real-life situation. Functional assessment of the variant's pathogenicity and comparison with exon 15 double knock-outs is currently ongoing.
Conclusion
In conclusion, we generated a new PKD cellular model that can be easily exploited to reproduce some of the VUS variants identified, by clinical exome sequencing, in our cohort of ADPKD patients. Results obtained provide a proof-of-principle of the feasibility of this approach and allowed to identify selective read-outs (Figure 1B) to be used for a rapid screening to assess the phenotypic impact of specific PKD1 variants.
Abstract
Background and Aims
Renal cystic disease (RCD) includes a spectrum of disorders with heterogenous clinical presentation. Among RCD are autosomal dominant polycystic kidney disease (ADPKD), ...autosomal recessive polycystic kidney disease (ARPKD), ciliopathies, HNF1B-nephropathy, and congenital anomalies of the kidneys and of the urinary tract (CAKUT). Diagnosis is based on clinical criteria, yet, given the extreme genetic heterogeneity and phenotypic overlap among different conditions, confirmation by genetic testing is paramount for correct clinical management and recurrence risk assessment. Although genotype-phenotype correlations are scanty, recently, coexisting variants in the same gene or in multiple cystogenes have been associated with earlier and more severe renal phenotype. The aim of this study is to describe the clinical and molecular characteristics of pediatric patients with early onset of RCD and provide genotype-phenotype correlations.
Method
A retrospective study was conducted collecting patients with a prenatal or early-childhood ultrasound finding of renal hyper-echogenicity or cysts. All patients were followed at the Pediatric Nephrology Unit of the Città della Salute e della Scienza University Hospital of Torino, Italy. Genetic analyses were mostly performed at the Immunogenetic and Transplant Biology Unit and included clinical exome sequencing (CES), array-CGH, MLPA and single gene testing.
Results
65 patients we recruited, 42 males (64.6%) and 23 females (35.4%), mean age 8 ± 5.9 years (range 0-18). The most frequent clinical suspicion was ADPKD (n = 40, 61.5%), followed by CAKUT (n = 9, 13.8%), ARPKD (n = 8, 12.3%), ciliopathy and tuberous sclerosis complex (TSC) (n = 3, 4.6% for each condition), and medullary cystic disease (n = 2, 3.1%).
Overall, causative variants (C4/C5) were identified in 39 patients (60.0%), 18 (27.7%) were carriers of variants of uncertain significance (C3) and 8 (12.3%) were inconclusive. Among the 40 patients with a clinical suspicion of ADPKD, 23 received a conclusive genetic diagnosis with 20 (87%) variants in PKD1, 2 (8.7%) in PKD2, one in HNF1B and 14 carried C3 variants. All patients with a clinical suspicion of ARPKD received molecular confirmation and, notably, in one case we observed biallelic variants in PKD1. Among the 21 patients with PKD1 variants, 6 (28.6%) had multiple PKD1 variants and 2 (9.5%) carried an additional PKHD1 variant. TSC diagnosis was confirmed in all 3 cases and two of them (66.7%) carried a contiguous gene TSC2-PKD1 deletion. Two out of 3 (66.7%) patients with a clinical suspicion of ciliopathy carried biallelic variants in BBS10, in one case associated with an additional variant in PKD2. Four (44.4%) CAKUT cases were solved with 3 causative variants in HNF1B (75%) and one in PAX2 (25%), 2 cases (22.2%) carried C3 variants and 3 (33.3%) were inconclusive. Causative variants were de novo in 10 cases (25.6%).
Conclusion
Our data confirms that genetic evaluation is a powerful tool for the diagnosis of RCD, as witnessed by a detection rate of 60%. Although the clinical suspicion was confirmed in the majority of cases, one suspected ARPKD case carried two PKD1 variants and one suspected ADPKD case carried a whole HNF1B gene deletion, highlighting the need for extensive genetic analysis. Moreover, nine patients had multiple variants potentially contributing with additive effect to a more severe renal phenotype: 6 patients had two variants in PKD1, 2 patients with a PKD1 variant had an additional variant in PKHD1 and one patient had biallelic BBS10 variants plus a nonsense PKD2 variant. In this context, genetic analysis by CES permits simultaneous evaluation of all RCD associated genes, uncovering both multiple variants and clinical phenocopies. In conclusion, the CES driven genetic characterization of pediatric patients with renal cysts allows to avoid misdiagnosis, minimize diagnostic invasive investigations, set up correct follow-up, and define recurrence risk, with a time- and cost- efficient approach.
PDF
