Vertebral, Cardiac, Renal and Limb Defect Syndrome (VCRL), is a very rare congenital malformation syndrome. Pathogenic variants in HAAO (3-Hydroxyanthranilate 3,4-dioxygenase), NADSYN1 (NAD+ ...Synthetase-1) and KYNU (Kynureninase) have been identified in a handful of affected individuals. All three genes encode for enzymes essential for the NAD+ de novo synthesis pathway. Using Trio-Exome analysis and CGH array analysis in combination with long range PCR, we have identified a novel homozygous copy number variant (CNV) encompassing exon 5 of KYNU in an individual presenting with overlapping features of VCRL and Catel–Manzke Syndrome. Interestingly, only the mother, not the father carried the small deletion in a heterozygous state. High-resolution SNP array analysis subsequently delineated a maternal isodisomy of chromosome 2 (UPD2). Increased xanthurenic acid excretion in the urine confirmed the genetic diagnosis. Our findings confirm the clinical, genetic and metabolic phenotype of VCRL1, adding a novel functionally tested disease allele. We also describe the first patient with NAD+ deficiency disorder resulting from a UPD. Furthermore, we provide a comprehensive review of the current literature covering the genetic basis and pathomechanisms for VCRL and Catel–Manzke Syndrome, including possible phenotype/genotype correlations as well as genetic causes of hypoplastic left heart syndrome.
In contrast to adult-onset inflammatory bowel disease (IBD), where many genetic loci have been shown to be involved in complex disease etiology, early-onset IBD (eoIBD) and associated syndromes can ...sometimes present as monogenic conditions. As a result, the clinical phenotype and ideal disease management in these patients often differ from those in adult-onset IBD. However, due to high costs and the complexity of data analysis, high-throughput screening for genetic causes has not yet become a standard part of the diagnostic work-up of eoIBD patients.
We selected 28 genes of interest associated with monogenic IBD and performed targeted panel sequencing in 71 patients diagnosed with eoIBD or early-onset chronic diarrhea to detect causative variants. We compared these results to whole-exome sequencing (WES) data available for 25 of these patients.
Target coverage was significantly higher in the targeted gene panel approach compared with WES, whereas the cost of the panel was considerably lower (approximately 25% of WES). Disease-causing variants affecting protein function were identified in 5 patients (7%), located in genes of the IL10 signaling pathway (3), WAS (1), and DKC1 (1). The functional effects of 8 candidate variants in 5 additional patients (7%) are under further investigation. WES did not identify additional causative mutations in 25 patients.
Targeted gene panel sequencing is a fast and effective screening method for monogenic causes of eoIBD that should be routinely established in national referral centers.
Hypomethylation of
and
can cause Silver-Russell syndrome (SRS), a clinically and genetically heterogeneous condition characterized by intrauterine growth restriction, poor postnatal growth, relative ...macrocephaly, craniofacial abnormalities, body asymmetry, hypoglycemia and feeding difficulties. Isolated hypomethylation of
has been reported in single cases of SRS as well. Here, we report on a 19-month-old patient who presented with two episodes of hypoglycemic seizures. No intrauterine growth restriction was observed, the patient did not present with SRS-typical facial features, and postnatal growth in the first months of life was along the lower normal percentiles. Exome sequencing did not reveal any likely pathogenic variants explaining the phenotype; however, hypomethylation studies revealed isolated hypomethylation of
, while the methylation of
appeared normal. Hypoglycemia responded well to growth hormone therapy, and the boy showed good catch-up growth. Our case demonstrates that SRS and isolated
hypomethylation should be considered early in the diagnosis of recurrent hypoglycemia in childhood, especially in combination with small gestational age and poor growth.
The release of extracellular traps (ETs) by granulocytes is a unique strategy to stop the dissemination of microbial pathogens. This study was undertaken to elucidate the potential of avian ...granulocytes (heterophils) to form ETs that can arrest and kill Salmonella organisms. After in vitro exposure of isolated heterophils and in vivo infection of day-old chicks with Salmonella enterica subsp. enterica serovars Infantis (SI) or Enteritidis (SE), the generation of ETs as well as the trapping and survivability of Salmonella organisms in the ET meshwork were determined by means of microscopy and spectrophotometry.
In vitro, heterophils were able to form ETs within 15min after SE and SI inoculation. At 120min and with a multiplicity of infection of 1 and 5, SI induced significantly more ETs and DNA release than SE. Both SE and SI were found to be associated with the ET structures. Live-dead staining showed most of the microorganisms within the extracellular scaffold alive.
In vivo, heterophils were detected in cecal lumen of SE-, but not SI-infected chicks. In cecum of the SE-exposed chicks, ET formations were scarcely detected whereas intact heterophils with phagocytosed bacteria were frequently found.
The results evidence the capability of heterophils to generate ETs after SE and SI exposure in vitro. However, an infection of chicks with Salmonella did not significantly induce the formation of ET structures in cecum. Thus, the process to form ETs (ETosis) seems not to be of special relevance for Salmonella defense within the cecal lumen of young chicks.
Spondylometaphyseal dysplasia Algerian type (MIM no.: 184253) is an uncommon autosomal dominant skeletal dysplasia caused by heterozygous mutations in the
gene (MIM no.: 120140). In this case based ...review, we reported a 5-year-old boy with short stature, severe dorsolumbar scoliosis, lumbar hyperlordosis, short trunk, and severe genu valgum
Radiological examination showed platyspondyly, irregular metaphyseal radiolucencies intermingled with radiodensities, and corner fractures. The patient has a c.3275G > A; p.Gly1092Asp mutation in exon 47 of the
gene and a variant of unknown significance in c.1366-13C > A in intron 21. This latter sequence variant could partially or completely disrupt the natural splice acceptor site of intron 21/exon 22 in the
gene leading to a potential modification of the phenotypic severity.
Dyskeratosis congenita (DC) is a rare telomere disease with pleiotropic manifestations and bone marrow failure (BMF) as a major cause of mortality. The genes involved in DC pathogenesis play a role ...in telomere maintenance but their function in other in biological systems has not been well characterized. Compound heterozygous missense mutations in TCAB1(WRAP53) were reported so far in two pedigrees with classic DC (Zhong et al, 2011). TCAB1 functions as a scaffold protein recruiting TERC to the sub-organelle Cajal bodies, thus contributing to the assembly of telomerase enzyme; among its other functions it regulates DNA double-strand break repair. Here we functionally characterize novel TCAB1 mutations and describe transcriptome and proteome profiles in patient-derived lymphoblastoid cell lines (LCL). Among a cohort of 50 DC patients we identified three pedigrees with TCAB1 mutations, BMF and telomeres below first percentile. Index 1 carried compound heterozygous mutations R155X/Y345C also present in the affected brother. Both siblings suffered from transfusion-dependent pancytopenia manifesting in the childhood and typical dyskeratotic features. Index 2 harbored a single mutation Q7TfsX27 and presented with hypocellular BMF. Although non-hematologic symptoms were initially absent, the patient suffered from mild restrictive lung disease after stem cell transplantation. Index 3 carried biallelic mutations Y345C/G435R. He was diagnosed with microcephaly, cerebellar hypoplasia, early onset BMF with immunodeficiency, consistent with Hoyeraal-Hreidarsson syndrome (HHS).
To gain insight into the functional consequences of novel mutations identified, we first assessed RNA, protein levels and localization in patient-derived LCLs and HeLa cells stably expressing wildtype (WT) and mutant TCAB1. The truncating mutations R155X and Q7TfsX27 were unable to translate to protein with abrogative localization in Cajal bodies, while the R155X mutation affected RNA stability through nonsense-mediated decay. The Y345C mutant was translated, but it was severely diminished in the nucleus and demonstrated defective localization in the Cajal bodies. The decline of TCAB1 signal in Cajal bodies was also observed in patients 1&3 with biallelic mutations.
Next, to describe the effect of mutations on cellular processes we performed RNA-sequencing (RNAseq) and mass spectrometric-based quantitative proteomics using SILAC on LCLs from patients/carriers and WT controls. The principal component analysis (PCA) of RNAseq data clusters the transcriptomes of patients with missense mutations (Y345C/G435R, Y345C) and truncating mutations (R155X, Q7TfsX27) into separate groups, while index 1 (R155X/Y345C) demonstrated a very distinct transcriptomic profile (Fig 1A). The telomere maintenance processes were highly downregulated for index 1 and clinically healthy carrier parents (Fig 1B). This might suggest that heterozygous mutations also affect telomere biology; however the compensatory effect from the WT allele allows maintaining normal homeostasis. Based on disease severity and established pathogenic effect of mutations we then focused on index patients 1 and 3 carrying biallelic mutations. The differential gene expression analysis suggested RPSA, LILRB1 (downregulated) and SH3BP5, TSPYL5 (upregulated) as top candidates. Strikingly, the identification of downregulated RPSA points at dysregulated ribosomal function as a possible novel mechanism in TCAB1-mutated cells. Finally, positive correlation was observed for differential gene regulation on transcriptome and protein level. A gene set variation analysis indicated down-regulation of telomere maintenance, protein processing and ubiquitination. Surprisingly the members of the telomerase holoenzyme complex were enriched on the protein level however they were detected transcriptionally down-regulated in patients (Fig 1C). This might hint at defective protein transport or slower protein turnover due to decreased ubiquitination.
In summary, we expand the clinical spectrum of TCAB1-associated DC ranging from BMF without mucocutaneous symptoms to severe HHS. We provide additional insights into the underlying cellular effects of TCAB1 mutations. The preliminary insight into the gene networks affected by TCAB1 mutations allows for first comprehension into the pleiotropic effects observed in patients.
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No relevant conflicts of interest to declare.
Abstract 1267
Diamond Blackfan anemia (DBA) has been established as a ribosomopathy and results in the majority of cases from the haploinsufficiency of genes coding for ribosomal proteins (RP). So ...far, 12 cytoplasmic RP genes (S7, S10, S17, S19, S24, S26, L5, L9, L11, L19, L26, and L35a) and the transcription factor GATA1 were identified to be affected in approximately half of all patients. Additionally, RP-gene deletions might constitute the causative mechanism. From the patients currently enrolled in the DBA registry in Germany, material for extensive genetic studies was available in 186 cases. We identified mutations in 52% (n=96) of patients in 9 of the before-mentioned 12 RP-genes. In addition, we investigated GATA1 and KLF1, a key erythroid transcription factor associated with the persistence of high levels of fetal hemoglobin (HbF), but no mutations in DBA patients were found. We next focused on the identification of RP-gene deletions within the mutation-negative cohort. Using Affymetrix 6.0 SNP-arrays and a qPCR-based copy number assay, we identified RP-gene deletions in 10% (n=19) patients, with S17 being the most frequently deleted gene in 6 patients, followed by L5, L11, L35a, and S19.
To assess whether the not yet identified genetic defects in the remaining ∼1/3 negative patients might have a different impact on the physical development and the clinical course than the RP-genes already linked to DBA, we compared both mutation/deletion positive (mut/del+) and negative cohorts. The age at diagnosis of mut/del+ patients versus negative patient group was 0.3 years as compared to 0.7 years, and there was equal sex ratio in both groups. HbF and eADA, used as lab surrogate markers, were increased in 73% mut/del+ as compared to 75% negative cases. The prevalence of physical abnormalities between both groups also seems to be similar. Specifically, no difference was seen between mut/del+ and negative patients for heart defects (37.5% and 26.5%), urogenital malformations (6.7% and 8.8%), thumb malformations (11.5% and 5.9%), small for gestational age (17% and 10.5%) and craniofacial malformations (39.4% and 35.3%). However cleft palate was exclusive to only one of 68 evaluable negative patients as compared to 6 of 104 evaluable mut/del+ positive patients. Interestingly, nearly all patients (94%) with gene deletions presented with physical anomalies, which might result from haploinsufficiency of other genes affected in the context of a continuous gene syndrome. We next focused our analysis on treatment modalities. Comparing the rate of spontaneous remission (SR), there were significantly more patients with SR within negative patient group as compared to mut/del+ cohort: 31.6% (18 of 57 evaluable) vs. 15.2% (15 of 99 evaluable), p < 0.04. Contrasting this, there was no difference for other treatment modalities including steroid/transfusion dependency.
In an attempt to identify whether the patients with an unknown genetic defect have impaired ribosome biogenesis, we performed polysomal profiling of 3 subgroups of DBA patients, those with point mutations in known RP genes, those with large deletions spanning known RP genes, and those that were deletion and mutation negative. Our preliminary results obtained from a cohort of 30 patients cell lines revealed not only the expected imbalance in 40S to 60S peak size depending on which RP gene was mutated, but also the presence of ribosomal intermediate structures in negative patients which were previously not reported in context of DBA. Some of these profiles with intermediates are linked to RP genes that have known links to rRNA processing. Interestingly, some of the profiles of the known RP genes were recapitulated in the profiles from patients with unknown genetic defect, suggesting that ribosomal profiling may be a valuable method of prediction for future DBA genotyping efforts.
In summary, DBA patients with unkown genetic defect show similar clinical presentation as patients with known RP-gene mutations or deletions. The observation of different treatment outcomes requires further investigation. Polysome profilling provides initial suggestion about the type of RP-defect and can help in better characterizing the patients prior to prospective next generation sequencing - based gene discovery.
No relevant conflicts of interest to declare.
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