Mono-allelic KCNB2 variants lead to a neurodevelopmental syndrome caused by altered channel inactivation.

Ion channels mediate voltage fluxes or action potentials that are central to the functioning of excitable cells such as neurons. The KCNB family of voltage-gated potassium channels (Kv) consists of two members (KCNB1 and KCNB2) encoded by KCNB1 and KCNB2, respectively. These channels are major contributors to delayed rectifier potassium currents arising from the neuronal soma which modulate overall excitability of neurons. In this study, we identified several mono-allelic pathogenic missense variants in KCNB2, in individuals with a neurodevelopmental syndrome with epilepsy and autism in some individuals. Recurrent dysmorphisms included a broad forehead, synophrys, and digital anomalies. Additionally, we selected three variants where genetic transmission has not been assessed, from two epilepsy studies, for inclusion in our experiments. We characterized channel properties of these variants by expressing them in oocytes of Xenopus laevis and conducting cut-open oocyte voltage clamp electrophysiology. Our datasets indicate no significant change in absolute conductance and conductance-voltage relationships of most disease variants as compared to wild type (WT), when expressed either alone or co-expressed with WT-KCNB2. However, variants c.1141A>G (p.Thr381Ala) and c.641C>T (p.Thr214Met) show complete abrogation of currents when expressed alone with the former exhibiting a left shift in activation midpoint when expressed alone or with WT-KCNB2. The variants we studied, nevertheless, show collective features of increased inactivation shifted to hyperpolarized potentials. We suggest that the effects of the variants on channel inactivation result in hyper-excitability of neurons, which contributes to disease manifestations.

Macrocephaly and developmental delay caused by missense variants in RAB5C.

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway.

Increased dynamin expression precedes proteinuria in glomerular disease.

Dynamin plays an essential role in maintaining the structure and function of the glomerular filtration barrier. Specifically, dynamin regulates the actin cytoskeleton and the turnover of nephrin in podocytes, and knocking down dynamin expression causes proteinuria. Moreover, promoting dynamin oligomerization with Bis-T-23 restores podocyte function and reduces proteinuria in several animal models of chronic kidney disease. Thus, dynamin is a promising therapeutic target for treating chronic kidney disease. Here, we investigated the pathophysiological role of dynamin under proteinuric circumstances in a rat model and in humans. We found that glomerular Dnm2 and Dnm1 mRNA levels are increased prior to the onset of proteinuria in a rat model of spontaneous proteinuria. Also, in zebrafish embryos, we confirm that knocking down dynamin translation results in proteinuria. Finally, we show that the glomerular expression of dynamin and cathepsin L protein is increased in several human proteinuric kidney diseases. We propose that the increased expression of glomerular dynamin reflects an exhausted attempt to maintain and/or restore integrity of the glomerular filtration barrier. These results confirm that dynamin plays an important role in maintaining the glomerular filtration barrier, and they support the notion that dynamin is a promising therapeutic target in proteinuric kidney disease. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Reply to Bouva et al. Comment on "Dijkstra et al. A False-Negative Newborn Screen for Tyrosinemia Type 1-Need for Re-Evaluation of Newborn Screening with Succinylacetone. Int. J. Neonatal Screen. 2023, 9, 66".

We thank the authors for their comments [...].

A False-Negative Newborn Screen for Tyrosinemia Type 1-Need for Re-Evaluation of Newborn Screening with Succinylacetone.

Undiagnosed and untreated tyrosinemia type 1 (TT1) individuals carry a significant risk for developing liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Elevated succinylacetone (SA) is pathognomonic for TT1 and therefore often used as marker for TT1 newborn screening (NBS). While SA was long considered to be elevated in every TT1 patient, here we present a recent false-negative SA TT1 screen. A nine-year-old boy presented with HCC in a cirrhotic liver. Additional tests for the underlying cause unexpectedly revealed TT1. Nine years prior, the patient was screened for TT1 via SA NBS with a negative result: SA 1.08 µmol/L, NBS cut-off 1.20 µmol/L. To our knowledge, this report is the first to describe a false-negative result from the TT1 NBS using SA. False-negative TT1 NBS results may be caused by milder TT1 variants with lower SA excretion. Such patients are more likely to be missed in NBS programs and can be asymptomatic for years. Based on our case, we advise TT1 to be considered in patients with otherwise unexplained liver pathology, including fibrosis, cirrhosis and HCC, despite a previous negative TT1 NBS status. Moreover, because the NBS SA concentration of this patient fell below the Dutch cut-off value (1.20 µmol/L at that time), as well as below the range of cut-off values used in other countries (1.29-10 µmol/L), it is likely that false-negative screening results for TT1 may also be occurring internationally. This underscores the need to re-evaluate TT1 SA NBS programs.

A Delphi Survey Study to Formulate Statements on the Treatability of Inherited Metabolic Disorders to Decide on Eligibility for Newborn Screening.

The Wilson and Jungner (W&J) and Andermann criteria are meant to help select diseases eligible for population-based screening. With the introduction of next-generation sequencing (NGS) methods for newborn screening (NBS), more inherited metabolic diseases (IMDs) can technically be included, and a revision of the criteria was attempted. This study aimed to formulate statements and investigate whether those statements could elaborate on the criterion of treatability for IMDs to decide on eligibility for NBS. An online Delphi study was started among a panel of Dutch IMD experts (EPs). EPs evaluated, amended, and approved statements on treatability that were subsequently applied to 10 IMDs. After two rounds of Delphi, consensus was reached on 10 statements. Application of these statements selected 5 out of 10 IMDs proposed for this study as eligible for NBS, including 3 IMDs in the current Dutch NBS. The statement: 'The expected benefit/burden ratio of early treatment is positive and results in a significant health outcome' contributed most to decision-making. Our Delphi study resulted in 10 statements that can help to decide on eligibility for inclusion in NBS based on treatability, also showing that other criteria could be handled in a comparable way. Validation of the statements is required before these can be applied as guidance to authorities.

Biochemical Studies in Fibroblasts to Interpret Variants of Unknown Significance in the ABCD1 Gene.

Due to newborn screening for X-linked adrenoleukodystrophy (ALD), and the use of exome sequencing in clinical practice, the detection of variants of unknown significance (VUS) in the ABCD1 gene is increasing. In these cases, functional tests in fibroblasts may help to classify a variant as (likely) benign or pathogenic. We sought to establish reference ranges for these tests in ALD patients and control subjects with the aim of helping to determine the pathogenicity of VUS in ABCD1. Fibroblasts from 36 male patients with confirmed ALD, 26 healthy control subjects and 17 individuals without a family history of ALD, all with an uncertain clinical diagnosis and a VUS identified in ABCD1, were included. We performed a combination of tests: (i) a test for very-long-chain fatty acids (VLCFA) levels, (ii) a D3-C22:0 loading test to study the VLCFA metabolism and (iii) immunoblotting for ALD protein. All ALD patient fibroblasts had elevated VLCFA levels and a reduced peroxisomal ß-oxidation capacity (as measured by the D3-C16:0/D3-C22:0 ratio in the D3-C22:0 loading test) compared to the control subjects. Of the VUS cases, the VLCFA metabolism was not significantly impaired (most test results were within the reference range) in 6/17, the VLCFA metabolism was significantly impaired (most test results were within/near the ALD range) in 9/17 and a definite conclusion could not be drawn in 2/17 of the cases. Biochemical studies in fibroblasts provided clearly defined reference and disease ranges for the VLCFA metabolism. In 15/17 (88%) VUS we were able to classify the variant as being likely benign or pathogenic. This is of great clinical importance as new variants will be detected.

Local dystrophin restoration with antisense oligonucleotide PRO051.

BACKGROUND: Duchenne's muscular dystrophy is associated with severe, progressive muscle weakness and typically leads to death between the ages of 20 and 35 years. By inducing specific exon skipping during messenger RNA (mRNA) splicing, antisense compounds were recently shown to correct the open reading frame of the DMD gene and thus to restore dystrophin expression in vitro and in animal models in vivo. We explored the safety, adverse-event profile, and local dystrophin-restoring effect of a single, intramuscular dose of an antisense oligonucleotide, PRO051, in patients with this disease. METHODS: Four patients, who were selected on the basis of their mutational status, muscle condition, and positive exon-skipping response to PRO051 in vitro, received a dose of 0.8 mg of PRO051 injected into the tibialis anterior muscle. A biopsy was performed 28 days later. Safety measures, composition of mRNA, and dystrophin expression were assessed. RESULTS: PRO051 injection was not associated with clinically apparent adverse events. Each patient showed specific skipping of exon 51 and sarcolemmal dystrophin in 64 to 97% of myofibers. The amount of dystrophin in total protein extracts ranged from 3 to 12% of that found in the control specimen and from 17 to 35% of that of the control specimen in the quantitative ratio of dystrophin to laminin alpha2. CONCLUSIONS: Intramuscular injection of antisense oligonucleotide PRO051 induced dystrophin synthesis in four patients with Duchenne's muscular dystrophy who had suitable mutations, suggesting that further studies might be feasible.

Selective loss of podoplanin protein expression accompanies proteinuria and precedes alterations in podocyte morphology in a spontaneous proteinuric rat model.

To evaluate changes during the development of proteinuria, podocyte morphology and protein expression were evaluated in spontaneously proteinuric, Dahl salt-sensitive (Dahl SS) rats. Dahl SS rats on a low-salt diet were compared with spontaneously hypertensive rats (SHR) at age 2, 4, 6, 8, and 10 weeks. Blood pressure, urinary protein excretion, urinary albumin excretion, and podocyte morphology were evaluated. In addition, the expression of 11 podocyte-related proteins was determined by analyzing protein and mRNA levels. In Dahl SS rats, proteinuria became evident around week 5, increasing thereafter. SHR rats remained non-proteinuric. Dahl SS rats showed widespread foot process effacement at 10 weeks. At < or =8 weeks, expression and distribution of the podocyte proteins was similar between the two strains, except for the protein podoplanin. At 4 weeks, podoplanin began decreasing in the glomeruli of Dahl SS rats in a focal and segmental fashion. Podoplanin loss increased progressively and correlated with albuminuria (r = 0.8, P < 0.001). Double labeling experiments revealed increased expression of the podocyte stress marker desmin in glomerular areas where podoplanin was lost. Dahl SS rats did not show podoplanin gene mutations or decreased mRNA expression. Thus, podocyte morphology and the expression and distribution of most podocyte-specific proteins were normal in young Dahl SS rats, despite marked proteinuria. Our study suggests that decreased expression of podoplanin plays a role in the decrease of glomerular permselectivity.

Genetic defect of the sodium-dependent multivitamin transporter: A treatable disease, mimicking biotinidase deficiency.

The sodium-dependent multivitamin transporter that facilitates the uptake of the water-soluble vitamins biotin, pantothenic acid, and the vitamin-like substance lipoate is coded by the SLC5A6 gene. Variants in this gene cause a relatively novel treatable metabolic disorder. Here we describe the second case. A 17-month-old girl presented with hypoglycemia (2.0 mmol/L) and severe metabolic acidosis (pH 6.87), leading to resuscitation. Her history revealed feeding problems from birth and poor weight gain. Metabolic investigation showed elevated plasma C3-carnitine and C5-OH-carnitine. Urine analysis showed persistently elevated excretion of 3-OH-isovaleric acid. Biochemically, the combination of elevated C5-OH-carnitine and increased excretion of 3-OH-isovaleric acid seemed compatible with biotinidase deficiency. Supplementation with biotin was started. Biotinidase activity in plasma showed only marginally decreased activity, which was considered insufficient explanation for her clinical symptoms. Subsequent trio-based whole exome sequencing revealed compound heterozygosity for variants in the SLC5A6 gene. Upon increasing the dosage of biotin supplementation and introduction of pantothenic acid supplementation, a striking clinical improvement was seen.

[Fever with a rash caused by chronic meningococcemia]. / Koorts en huiduitslag door chronische meningokokkemie.

BACKGROUND: Fever with a rash is a common clinical presentation, which can be caused by various medical conditions. CASE DESCRIPTION: A 14-year old boy presented at the outpatient clinic with a two-week history of fever, myalgia and purpuric skin lesions. Blood cultures showed an infection with Neisseria meningitidis. After antibiotic treatment, his symptoms resolved promptly. CONCLUSION: Chronic meningococcemia is a rare manifestation of meningococcal infection and should be considered in patients with prolonged fever, purpuric skin lesions and joint involvement.

[A neonate with peculiarly arranged skin abnormalities]. / Een baby met bijzonder gerangschikte huidafwijkingen.

A tropical doctor reviewed a female Malawian neonate with a vesicular rash and linear hyperpigmentation that followed the lines of Blaschko. This distribution is typical for incontinentia pigmenti, a congenital X-linked dermatosis, which usually resolves spontaneously in adolescence.

Pellagra: a non-communicable disease of poverty.

Pellagra is a disease of poverty that is treatable but easily overlooked. The disease is caused by a deficiency of niacin and is clinically characterized by the triad of dermatitis, diarrhoea and dementia. We describe two cases of pellagra in a Malawian district hospital and discuss the pathogenesis, clinical manifestations and treatment of the disease.

C4d staining in renal allograft biopsies with early acute rejection and subsequent clinical outcome.

BACKGROUND AND OBJECTIVES: Diffuse C4d staining in peritubular capillaries (PTCs) during an acute rejection episode (ARE) is the footprint of antibody-mediated rejection. In current clinical practice, diffuse C4d+ staining during acute rejection is regarded as an inferior prognostic sign. This case-control study investigated the prognostic role of mere C4d staining for graft outcome during an ARE in a well defined cohort of similarly ARE-treated patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: All kidney transplant recipients in the authors' center from January 1, 1995 to December 31, 2005 were reviewed. From these patients, 151 had a clinical ARE. Paraffin and/or frozen material was available for 128 patients showing a histologically proven ARE within the first 6 months after transplantation. All ARE patients were treated similarly with high-dose pulse steroids and in the case of steroid unresponsiveness with anti-thymocyte globulin. Biopsies were scored according to Banff criteria. Frozen and paraffin sections were stained by immunofluorescence (IF) and immunohistochemistry (IHC) for C4d, respectively, and scored for PTC positivity. RESULTS: Diffuse C4d+ staining in PTCs was found in 12.5% and 4.2% sections stained by IF or by IHC, respectively. Four patients showed diffuse positive staining with both methods but showed no different risk profile from other patients. No relation between C4d staining and clinical parameters at baseline was found. C4d staining was not associated with steroid responsiveness, graft, or patient survival. CONCLUSIONS: This study shows that C4d staining is not related to clinical outcome in this cohort of histologically proven early AREs.