Proteomic and bioinformatic profiling of neutrophils in CLL reveals functional defects that predispose to bacterial infections.

Patients with chronic lymphocytic leukemia (CLL) typically suffer from frequent and severe bacterial infections. Although it is well known that neutrophils are critical innate immune cells facilitating the early defense, the underlying phenotypical and functional changes in neutrophils during CLL remain largely elusive. Using a murine adoptive transfer model of CLL, we demonstrate aggravated bacterial burden in CLL-bearing mice upon a urinary tract infection with uropathogenic Escherichia coli. Bioinformatic analyses of the neutrophil proteome revealed increased expression of proteins associated with interferon signaling and decreased protein expression associated with granule composition and neutrophil migration. Functional experiments validated these findings by showing reduced levels of myeloperoxidase and acidification of neutrophil granules after ex vivo phagocytosis of bacteria. Pathway enrichment analysis indicated decreased expression of molecules critical for neutrophil recruitment, and migration of neutrophils into the infected urinary bladder was significantly reduced. These altered migratory properties of neutrophils were also associated with reduced expression of CD62L and CXCR4 and correlated with an increased incidence of infections in patients with CLL. In conclusion, this study describes a molecular signature of neutrophils through proteomic, bioinformatic, and functional analyses that are linked to a reduced migratory ability, potentially leading to increased bacterial infections in patients with CLL.

Loss of Functional Osteoprotegerin: More Than a Skeletal Problem.

Introduction: Juvenile Paget's disease (JPD), an ultra-rare, debilitating bone disease due to loss of functional osteoprotegerin (OPG), is caused by recessive mutations in TNFRFSF11B. A genotype-phenotype correlation spanning from mild to very severe forms is described. Aim: This study aimed to describe the complexity of the human phenotype of OPG deficiency in more detail and to investigate heterozygous mutation carriers for clinical signs of JPD. Patients: We investigated 3 children with JPD from families of Turkish, German, and Pakistani descent and 19 family members (14 heterozygous). Results: A new disease-causing 4 bp-duplication in exon 1 was detected in the German patient, and a microdeletion including TNFRFSF11B in the Pakistani patient. Skeletal abnormalities in all affected children included bowing deformities and fractures, contractures, short stature and skull involvement. Complex malformation of the inner ear and vestibular structures (2 patients) resulted in early deafness. Patients were found to be growth hormone deficient (2), displayed nephrocalcinosis (1), and gross motor (3) and mental (1) retardation. Heterozygous family members displayed low OPG levels (12), elevated bone turnover markers (7), and osteopenia (6). Short stature (1), visual impairment (2), and hearing impairment (1) were also present. Conclusion: Diminished OPG levels cause complex changes affecting multiple organ systems, including pituitary function, in children with JPD and may cause osteopenia in heterozygous family members. Diagnostic and therapeutic measures should aim to address the complex phenotype.

Effect of propofol in the immature rat brain on short- and long-term neurodevelopmental outcome.

BACKGROUND: Propofol is commonly used as sedative in newborns and children. Recent experimental studies led to contradictory results, revealing neurodegenerative or neuroprotective properties of propofol on the developing brain. We investigated neurodevelopmental short- and long-term effects of neonatal propofol treatment. METHODS: 6-day-old Wistar rats (P6), randomised in two groups, received repeated intraperitoneal injections (0, 90, 180 min) of 30 mg/kg propofol or normal saline and sacrificed 6, 12 and 24 hrs following the first injection. Cortical and thalamic areas were analysed by Western blot and quantitative real-time PCR (qRT-PCR) for expression of apoptotic and neurotrophin-dependent signalling pathways. Long-term effects were assessed by Open-field and Novel-Object-Recognition at P30 and P120. RESULTS: Western blot analyses revealed a transient increase of activated caspase-3 in cortical, and a reduction of active mitogen-activated protein kinases (ERK1/2, AKT) in cortical and thalamic areas. qRT-PCR analyses showed a down-regulation of neurotrophic factors (BDNF, NGF, NT-3) in cortical and thalamic regions. Minor impairment in locomotive activity was observed in propofol treated adolescent animals at P30. Memory or anxiety were not impaired at any time point. CONCLUSION: Exposing the neonatal rat brain to propofol induces acute neurotrophic imbalance and neuroapoptosis in a region- and time-specific manner and minor behavioural changes in adolescent animals.

Effects of RANK-ligand antibody (denosumab) treatment on bone turnover markers in a girl with juvenile Paget's disease.

CONTEXT: Juvenile Paget's disease (JPD) is an extremely rare, yet painful and debilitating bone disease with onset occurring during early childhood. JPD can be caused by loss of function of osteoprotegerin, resulting in subsequent stimulation of osteoclasts via the receptor activator of nuclear factor-κB (RANK) pathway. Increased bone turnover and lack of bone modeling lead to severe deformities, frequent fractures, short stature, and loss of hearing. SETTING: The treatment for JPD is challenging and has previously been based on administration of either calcitonin or bisphosphonates. However, with the development of denosumab, a receptor activator of nuclear factor-κB-ligand (RANKL) antibody, a treatment targeting the pathophysiology of JPD may be available. We report the effects of denosumab treatment on an 8-year-old girl with a severe form of JPD. PATIENT: Before starting the denosumab treatment regimen, the patient had been treated for 3.5 years with iv pamidronate. INTERVENTION AND OUTCOME: The administration of denosumab resulted in improved disease control compared with bisphosphonate, as assessed by monitoring markers of bone turnover. Alkaline phosphatase levels dropped within the normal range and remained at normal levels for 5 months after the final dose of denosumab. Additionally, bone pain was more efficiently controlled with denosumab. However, concomitant with the first injection, severe hypocalcemia developed, for which the patient was hospitalized and iv calcium supplementation was required for 13 days. CONCLUSIONS: Denosumab appears to be significantly effective for osteoclast inhibition for the treatment of JPD. However, in our patient, denosumab administration was associated with severe hypocalcemia, indicating that close monitoring of calcium levels is required during treatment.

Congenital myasthenic syndrome with tubular aggregates caused by GFPT1 mutations.

Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous group of inherited disorders of the neuromuscular junction. A difficult to diagnose subgroup of CMS is characterised by proximal muscle weakness and fatigue while ocular and facial involvement is only minimal. DOK7 mutations have been identified as causing the disorder in about half of the cases. More recently, using classical positional cloning, we have identified mutations in a previously unrecognised CMS gene, GFPT1, in a series of DOK7-negative cases. However, detailed description of clinical features of GFPT1 patients has not been reported yet. Here we describe the clinical picture of 24 limb-girdle CMS (LG-CMS) patients and pathological findings of 18 of them, all carrying GFPT1 mutations. Additional patients with CMS, but without tubular aggregates, and patients with non-fatigable weakness with tubular aggregates were also screened. In most patients with GFPT1 mutations, onset of the disease occurs in the first decade of life with characteristic limb-girdle weakness and fatigue. A common feature was beneficial and sustained response to acetylcholinesterase inhibitor treatment. Most of the patients who had a muscle biopsy showed tubular aggregates in myofibers. Analysis of endplate morphology in one of the patients revealed unspecific abnormalities. Our study delineates the phenotype of CMS associated with GFPT1 mutations and expands the understanding of neuromuscular junction disorders. As tubular aggregates in context of a neuromuscular transmission defect appear to be highly indicative, we suggest calling this condition congenital myasthenic syndrome with tubular aggregates (CMS-TA).

Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect.

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.

Expression of SNURF-SNRPN upstream transcripts and epigenetic regulatory genes during human spermatogenesis.

The imprinted domain in human 15q11-q13 is controlled by a bipartite imprinting centre (IC), which overlaps the 5' part of the paternally expressed SNURF-SNRPN gene. We have recently described two novel genes upstream of SNURF-SNRPN (PWRN1 and PWRN2), which are biallelically expressed in the testis. We have now found that PWRN1 represents an alternative 5' part of SNURF-SNRPN, and that its expression in the brain is imprinted. To determine when the locus is activated during spermatogenesis and which factors are involved in this process, we have mined gene-expression data of testicular biopsies from men with different types of spermatogenic failure. Whereas PWRN1-SNURF-SNRPN and PWRN2 are expressed in post-meiotic germ cells only, a hitherto undetected SNURF-SNRPN upstream transcript is expressed already at meiosis. Several epigenetic factors (eg, MBD1 and MBD2 isoforms, MBD3L1, SUVH39H2, BRDT, and EZH2) are upregulated at specific stages of spermatogenesis, suggesting that they play an important role in the epigenetic reprogramming during spermatogenesis.

Mutations in the slow skeletal muscle fiber myosin heavy chain gene (MYH7) cause laing early-onset distal myopathy (MPD1).

We previously linked Laing-type early-onset autosomal dominant distal myopathy (MPD1) to a 22-cM region of chromosome 14. One candidate gene in the region, MYH7, which is mutated in cardiomyopathy and myosin storage myopathy, codes for the myosin heavy chain of type I skeletal muscle fibers and cardiac ventricles. We have identified five novel heterozygous mutations--Arg1500Pro, Lys1617del, Ala1663Pro, Leu1706Pro, and Lys1729del in exons 32, 34, 35, and 36 of MYH7--in six families with early-onset distal myopathy. All five mutations are predicted, by in silico analysis, to locally disrupt the ability of the myosin tail to form the coiled coil, which is its normal structure. These findings demonstrate that heterozygous mutations toward the 3' end of MYH7 cause Laing-type early-onset distal myopathy. MYH7 is the fourth distal-myopathy gene to have been identified.

Phenotypic spectrum associated with mutations in the fukutin-related protein gene.

We describe 22 patients with mutations in the fukutin-related protein (FKPR) gene. Four patients had congenital muscular dystrophy (MDC1C), with presentation at birth, severe weakness and inability to stand unsupported. The other 18 had limb girdle muscular dystrophy (LGMD2I). Eleven showed a Duchenne-like course with loss of ambulation in the early teens while 7 had a milder phenotype. Muscle biopsy invariably showed abnormal expression of a-dystroglycan. MDC1C patients either carried 2 missense or 1 missense and 1 nonsense mutations. Patients with LGMD2I shared a common mutation (C826A,Leu276Ileu) and their phenotypic severity was correlated with the second allelic mutation.