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BACKGROUND Over the last three decades, technological developments facilitating assisted reproductive techniques (ART) have revolutionized the treatment of subfertile couples, including men suffering from severe oligospermia or azoospermia. In parallel with the advent of these technologies, there is a great concern about the biological safety of ART. This concern is supported by the clinical observation that the frequency of congenital malformations is slightly elevated among ART-conceived children. METHODS In this explorative study, we have used tiling-resolution BAC array-mediated comparative genomic hybridization to investigate the incidence of de novo genomic copy number changes in a group of 12 ICSI children, compared with a control group of 30 naturally conceived children. RESULTS In 6 of the 12 ICSI children, we found 10 apparently de novo ‘same direction genomic copy number changes’ [i.e. simultaneous copy number gain (or loss) with respect to both biological parents], notably losses. In statistically significant contrast, similar observations were encountered only six times in the control group in 5 of the 30 children. However, our study group was small, so a larger group is needed to confirm these findings. CONCLUSIONS Loci at which we found de novo alterations are known from the human genome database to be prone to large DNA segment copy number changes. As discussed, various molecular mechanisms, including the consequences of delayed male meiotic synapsis and replication fork stalling at early embryonic cell cycles, might trigger these copy number changes. [ABSTRACT FROM AUTHOR]

Several known or putative glycosyltransferases are required for the synthesis of laminin-binding glycans on alpha-dystroglycan (αDG), including POMT1, POMT2, POMGnT1, LARGE, Fukutin, FKRP, ISPD and GTDC2. Mutations in these glycosyltransferase genes result in defective αDG glycosylation and reduced ligand binding by αDG causing a clinically heterogeneous group of congenital muscular dystrophies, commonly referred to as dystroglycanopathies. The most severe clinical form, Walker-Warburg syndrome (WWS), is characterized by congenital muscular dystrophy and severe neurological and ophthalmological defects. Here, we report two homozygous missense mutations in the β-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) gene in a family affected with WWS. Functional studies confirmed the pathogenicity of the mutations. First, expression of wild-type but not mutant B3GNT1 in human prostate cancer (PC3) cells led to increased levels of αDG glycosylation. Second, morpholino knockdown of the zebrafish b3gnt1 orthologue caused characteristic muscular defects and reduced αDG glycosylation. These functional studies identify an important role of B3GNT1 in the synthesis of the uncharacterized laminin-binding glycan of αDG and implicate B3GNT1 as a novel causative gene for WWS.

Walker-Warburg syndrome (WWS) is an autosomal recessive multisystem disorder characterized by complex eye and brain abnormalities with congenital muscular dystrophy (CMD) and aberrant a-dystroglycan glycosylation. Here we report mutations in the ISPD gene (encoding isoprenoid synthase domain containing) as the second most common cause of WWS. Bacterial IspD is a nucleotidyl transferase belonging to a large glycosyltransferase family, but the role of the orthologous protein in chordates is obscure to date, as this phylum does not have the corresponding non-mevalonate isoprenoid biosynthesis pathway. Knockdown of ispd in zebrafish recapitulates the human WWS phenotype with hydrocephalus, reduced eye size, muscle degeneration and hypoglycosylated a-dystroglycan. These results implicate ISPD in a-dystroglycan glycosylation in maintaining sarcolemma integrity in vertebrates.

Background: Familial neurohypophyseal (central) diabetes insipidus (DI) is caused by mutations in the arginine vasopressin-neurophysin II (AVP-NPII) gene. The majority of cases is inherited in an autosomal dominant way. In this study, we present the clinical features of a mother and her son with autosomal dominant neurohypophyseal DI caused by a novel mutation., Case: A thirty-four-year-old woman and her three-year-old son were evaluated because of polyuria and polydipsia since the age of 1.5 years onwards. Both patients were subjected to a water deprivation test confirming the diagnosis of central DI. Magnetic resonance imaging of the brain of the mother showed a hypothalamus without apparent abnormalities and a relatively small neurohypophysis without a hyperintense signal. Mutation analysis showed a c.322G>T (p.?/p.Glu108X) in Exon 2 of the AVP-NPII gene in both mother and son., Discussion: This study reports neurohypophyseal DI in a mother and her son due to a novel mutation in Exon 2 of the AVP-NPII gene. Clinical and pathophysiological aspects of this disease are shortly reviewed and discussed.

Dystroglycanopathies are a heterogeneous group of disorders caused by defects in the glycosylation pathway of alpha-dystroglycan. The clinical spectrum ranges from severe congenital muscular dystrophy with structural brain and eye involvement to a relatively mild adult onset limb-girdle muscular dystrophy without brain abnormalities and normal intelligence. Mutations have been identified in one of six putative or demonstrated glycosyltransferases. Many different FKRP mutations have been identified, which cover the complete clinical spectrum of dystroglycanopathies. In contrast to the other known genes involved in these disorders, genotype-phenotype correlations are not obvious for FKRP mutations. To date, no homozygous or compound heterozygous null mutations have been identified in FKRP, suggesting that null mutations in FKRP could result in embryonic lethality. We report a family with two siblings carrying a homozygous mutation in the start codon of FKRP that is likely to result in a loss of functional FKRP protein. The clinical phenotype of the patients was consistent with Walker-Warburg syndrome, the most severe disorder in the disease spectrum of dystroglycanopathies.

Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved. Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glycosylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of the three DPM subunits and complementation of DPM3-deficient CHO2.38 cells showed a pathogenic p.L85S missense mutation in the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycanopathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.

Background: Over the last three decades, technological developments facilitating assisted reproductive techniques (ART) have revolutionized the treatment of subfertile couples, including men suffering from severe oligospermia or azoospermia. In parallel with the advent of these technologies, there is a great concern about the biological safety of ART. This concern is supported by the clinical observation that the frequency of congenital malformations is slightly elevated among ART-conceived children., Methods: In this explorative study, we have used tiling-resolution BAC array-mediated comparative genomic hybridization to investigate the incidence of de novo genomic copy number changes in a group of 12 ICSI children, compared with a control group of 30 naturally conceived children., Results: In 6 of the 12 ICSI children, we found 10 apparently de novo 'same direction genomic copy number changes' [i.e. simultaneous copy number gain (or loss) with respect to both biological parents], notably losses. In statistically significant contrast, similar observations were encountered only six times in the control group in 5 of the 30 children. However, our study group was small, so a larger group is needed to confirm these findings., Conclusions: Loci at which we found de novo alterations are known from the human genome database to be prone to large DNA segment copy number changes. As discussed, various molecular mechanisms, including the consequences of delayed male meiotic synapsis and replication fork stalling at early embryonic cell cycles, might trigger these copy number changes.

The m.13513G > A transition in the mitochondrial gene encoding the ND5 subunit of respiratory chain complex I, can cause mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and has been reported to be a frequent cause of Leigh syndrome (LS). We determined the frequency of the mutation in a cohort of 123 patients with reduced complex I activity in muscle (n = 113) or fibroblast (n = 10) tissue. We describe a Pyrosequencing assay for rapid detection and quantification of the m.13513G > A mutation. Two patients with the mutation were identified; both had LS, optical atrophy and a Wolff-Parkinson-White Syndrome (WPWS)-like cardiac conduction defect. The clinical presentation of the m.13513G > A mutation is discussed. We conclude that the m.13513G > A mutation seems not as frequent as previously suggested and is most likely to be present in patients with Leigh (-like) syndrome combined with a complex I deficiency, optic atrophy and/ or WPWS. In addition, we confirmed that the adjacent m.13514A > G mutation is a rare cause of LS or MELAS since no cases with this transition were found.

Recognizing an ALS-mimic can be challenging. Here, we describe a patient with a slowly progressive dysarthria and dysphagia, with fasciculations of the tongue and general hyperreflexia, fulfilling the diagnostic criteria of 'clinical probable ALS'. Because of a non-conclusive EMG, a muscle biopsy was performed that surprisingly showed widespread nemaline rods. The clinical features and the histological findings were compatible with a sporadic late onset nemaline myopathy. Three years after initial presentation the patient died and post-mortem examination not only showed nemaline bodies in every muscle examined, but also revealed an unsuspected final diagnosis: sarcoid brainstem encephalitis. Nemaline rods can be found in various disorders, and neurosarcoidosis should be added to this list.

The importance of O-glycosylation of alpha-dystroglycan (alpha-DG) is evident from the identification of POMT1 mutations in Walker-Warburg syndrome (WWS). Approximately one-fifth of the WWS patients show mutations in POMT1, which result in complete loss of protein mannosyltransferase activity. WWS patients are characterized by congenital muscular dystrophy (CMD) with severe brain and eye abnormalities. This suggests a crucial role for alpha-DG during development of these organs and tissues. Here we report new POMT1 mutations and polymorphisms in WWS patients. In addition, we report different compound heterozygous POMT1 mutations in four unrelated families that result in a less severe phenotype than WWS, characterized by CMD with calf hypertrophy, microcephaly, and mental retardation. Compared to WWS patients, these patients have milder structural brain abnormalities, and eye abnormalities were absent, except for myopia in some cases. In these patients we postulate that one or both transcripts for POMT1 confer residual protein O-mannosyltransferase activity. Our data suggest the existence of a disease spectrum of CMD including brain and eye abnormalities resulting from POMT1 mutations., ((c) 2006 Wiley-Liss, Inc.)

Background: Walker-Warburg syndrome (WWS) is an autosomal recessive condition characterised by congenital muscular dystrophy, structural brain defects, and eye malformations. Typical brain abnormalities are hydrocephalus, lissencephaly, agenesis of the corpus callosum, fusion of the hemispheres, cerebellar hypoplasia, and neuronal overmigration, which causes a cobblestone cortex. Ocular abnormalities include cataract, microphthalmia, buphthalmos, and Peters anomaly. WWS patients show defective O-glycosylation of alpha-dystroglycan (alpha-DG), which plays a key role in bridging the cytoskeleton of muscle and CNS cells with extracellular matrix proteins, important for muscle integrity and neuronal migration. In 20% of the WWS patients, hypoglycosylation results from mutations in either the protein O-mannosyltransferase 1 (POMT1), fukutin, or fukutin related protein (FKRP) genes. The other genes for this highly heterogeneous disorder remain to be identified., Objective: To look for mutations in POMT2 as a cause of WWS, as both POMT1 and POMT2 are required to achieve protein O-mannosyltransferase activity., Methods: A candidate gene approach combined with homozygosity mapping., Results: Homozygosity was found for the POMT2 locus at 14q24.3 in four of 11 consanguineous WWS families. Homozygous POMT2 mutations were present in two of these families as well as in one patient from another cohort of six WWS families. Immunohistochemistry in muscle showed severely reduced levels of glycosylated alpha-DG, which is consistent with the postulated role for POMT2 in the O-mannosylation pathway., Conclusions: A fourth causative gene for WWS was uncovered. These genes account for approximately one third of the WWS cases. Several more genes are anticipated, which are likely to play a role in glycosylation of alpha-DG.

The potency of live attenuated virus vaccines is determined by counting or titrating viable viruses in cell cultures. These classical potency tests have the drawback that they are time consuming and laborious and show a high laboratory-to-laboratory variation. In the present study we describe the development and validation of a fast method to measure the potency of measles in trivalent measles, mumps and rubella (MMR) vaccines using quantitative real-time PCR (qPCR). Vero cells were infected with serial dilutions of a trivalent vaccine or a trivalent reference with known potency. Virus was allowed to replicate and subsequently replicated virus was quantitated by qPCR using the LightCycler technology. The virus titer in vaccine samples was estimated against reference preparations using parallel line analysis. In comparison to the plaque assay, the qPCR infectivity assay was faster and less laborious, while accuracy and intermediate precision were similar.