Validation of a method for the determination of triphenylmethane dyes in trout and shrimp with superior extraction efficiency.

The described methods are able to analyse the triphenylmethane dyes malachite green (MG), crystal violet (CV) and brilliant green (BG) as well as their leuco metabolites leuco malachite green (LMG), leuco crystal violet (LCV) and leuco brilliant green (LBG) on the basis of a simple and fast extraction. The validation of the methods in two studies without and with a heated ultrasonic treatment during the extraction of fortified trout and shrimp samples was successfully performed applying an in-house validation concept. The evaluation of the relevant validation parameters, e.g. the decision limit CCα, the detection capability CCß, the repeatability, the within-laboratory reproducibility and the recovery for both extraction versions, showed results which fulfil the requirements of Commission Decision 2002/657/EC. The investigation of incurred material of trout containing the above compounds with an additional heated ultrasonic treatment during extraction leads to higher findings of MG and BG. This effect was also confirmed by other laboratories in the framework of a proficiency test. For CV and all three leuco metabolites no increase in the detected amounts could be observed after a heated ultrasonic treatment of the incurred trout material.

Targeted next generation sequencing as a diagnostic tool in epileptic disorders.

PURPOSE: Epilepsies have a highly heterogeneous background with a strong genetic contribution. The variety of unspecific and overlapping syndromic and nonsyndromic phenotypes often hampers a clear clinical diagnosis and prevents straightforward genetic testing. Knowing the genetic basis of a patient's epilepsy can be valuable not only for diagnosis but also for guiding treatment and estimating recurrence risks. METHODS: To overcome these diagnostic restrictions, we composed a panel of genes for Next Generation Sequencing containing the most relevant epilepsy genes and covering the most relevant epilepsy phenotypes known so far. With this method, 265 genes were analyzed per patient in a single step. We evaluated this panel on a pilot cohort of 33 index patients with concise epilepsy phenotypes or with a severe but unspecific seizure disorder covering both sporadic and familial cases. KEY FINDINGS: We identified presumed disease-causing mutations in 16 of 33 patients comprising sequence alterations in frequently as well as in less commonly affected genes. The detected aberrations encompassed known and unknown point mutations (SCN1A p.R222X, p. E289V, p.379R, p.R393H; SCN2A p.V208E; STXBP1 p.R122X; KCNJ10 p.L68P, p.I129V; KCTD7 p.L108M; KCNQ3 p.P574S; ARHGEF9 p.R290H; SMS p.F58L; TPP1 p.Q278R, p.Q422H; MFSD8 p.T294K), a putative splice site mutation (SCN1A c.693A> p.T/P231P) and small deletions (SCN1A p.F1330Lfs3X [1 bp]; MFSD8 p.A138Dfs10X [7 bp]). All mutations have been confirmed by conventional Sanger sequencing and, where possible, validated by parental testing and segregation analysis. In three patients with either Dravet syndrome or myoclonic epilepsy, we detected SCN1A mutations (p.R222X, p.P231P, p.R393H), even though other laboratories had previously excluded aberrations of this gene by Sanger sequencing or high-resolution melting analysis. SIGNIFICANCE: We have developed a fast and cost-efficient diagnostic screening method to analyze the genetic basis of epilepsies. We were able to detect mutations in patients with clear and with unspecific epilepsy phenotypes, to uncover the genetic basis of many so far unresolved cases with epilepsy including mutation detection in cases in which previous conventional methods yielded falsely negative results. Our approach thus proved to be a powerful diagnostic tool that may contribute to collecting information on both common and unknown epileptic disorders and in delineating associated phenotypes of less frequently mutated genes.

Haploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly.

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EFTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the first multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.

Validation of a multi-residue method for the determination of several antibiotic groups in honey by LC-MS/MS.

The presented multi-method was developed for the confirmation of 37 antibiotic substances from the six antibiotic groups: macrolides, lincosamides, quinolones, tetracyclines, pleuromutilines and diamino-pyrimidine derivatives. All substances were analysed simultaneously in a single analytical run with the same procedure, including an extraction with buffer, a clean-up by solid-phase extraction, and the measurement by liquid chromatography tandem mass spectrometry in ESI+ mode. The method was validated on the basis of an in-house validation concept with factorial design by combination of seven factors to check the robustness in a concentration range of 5-50 µg kg(-1). The honeys used were of different types with regard to colour and origin. The values calculated for the validation parameters-decision limit CCα (range, 7.5-12.9 µg kg(-1)), detection capability CCß (range, 9.4-19.9 µg kg(-1)), within-laboratory reproducibility RSD(wR) (<20% except for tulathromycin with 23.5% and tylvalosin with 21.4 %), repeatability RSD(r) (<20% except for tylvalosin with 21.1%), and recovery (range, 92-106%)-were acceptable and in agreement with the criteria of Commission Decision 2002/657/EC. The validation results showed that the method was applicable for the residue analysis of antibiotics in honey to substances with and without recommended concentrations, although some changes had been tested during validation to determine the robustness of the method.

Preparation and characterisation of in-house reference material of tylosin in honey and results of a proficiency test.

The analysis of incurred material from animals treated with pharmacologically active substances is an efficient way to check the accuracy of a method. Tylosin A was chosen for the preparation of that material because it is highly effective in controlling active infections of American Foulbrood (AFB), a global threat to apiculture, but residues in honey are not allowed according to European legislation. For this reason an in-house reference material of honey containing the macrolide tylosin A and its degradation product desmycosin (tylosin B) was prepared. After the treatment of a beehive with the appropriate macrolide tylosin A, the honey samples were collected. The incurred honey material was diluted by mixing with blank honey. Concentrations of 25.81 µg kg(-1) for tylosin A and of 19.28 µg kg(-1) for its degradation product desmycosin (tylosin B) were reached. The homogeneity was checked by analysing 12 bottles in duplicate. The stability was tested at different defined temperatures and storage conditions. The reference material described above was homogeneous and stable. Samples of this in-house reference material were used for the realisation of a proficiency test with international participation. All participants accomplished satisfying results with the exception of one laboratory.

Validated determination of eight antibiotic substance groups in cattle and pig muscle by HPLC/MS/MS.

The described multimethod is suited for the determination of 53 substances of eight antibiotic groups-tetracyclines, quinolones, macrolides, sulfonamides, diphenylsulfones, diamino-pyrimidine derivatives, pleuromutilines, and lincosamides-in cattle and pig muscle. All substances were analyzed simultaneously with the same sample preparation and in one HPLC/MS/MS run. The validation of the multimethod was successfully accomplished with the help of an alternative in-house validation concept requiring only 48 experiments. The substances were validated at concentrations of 0.25, 0.5, 1.0, 1.5, and 2.0 x MRL (maximum residue limit) or 5, 10, 20, 30, and 40 microg/kg for substances without an MRL. The calculated relevant validation parameters were based on and comply with the requirements of Commission Decision 2002/657/EC, i.e., the decision limit, detection capability, repeatability, within-laboratory reproducibility, and recovery. The robustness of the method was demonstrated by varying seven factors of the analytical procedure. Several proficiency tests were carried out successfully to provide evidence for the applicability of the method.

An emerging 1q21.1 deletion-associated neurodevelopmental phenotype.

In this study, we describe the neurodevelopmental and epileptic phenotypes in a family with an inherited 1q21.1 deletion. During the pregnancy with the proband, increased nuchal translucency and oligohydramnion were detected. The proband showed mild global developmental delay and ataxic gait. Seizures started in the proband at the age of 2 years and manifested as generalized tonic-clonic seizures, atypical absence seizures, head drops, and drop attacks with no abnormal findings on interictal electroencephalogram. We performed an Agilent Human Genome CGH (comparative genomic hybridization) Microarray 105A, and a microdeletion on chromosome 1q21.1 was identified in both the patient and his asymptomatic father. This deletion encompasses 1.65 Mb and is larger than the reported recurrent class I deletions in this region. Cryptic cytogenetic abnormalities should be considered in patients with neurodevelopmental problems and atypical presentation of epilepsy with a normal electroencephalography (EEG).

Confirmatory method for the determination of streptomycin in apples by LC-MS/MS.

The method was specifically developed for the determination and confirmation of streptomycin in apple samples using the whole mellow apple. The method is simple, rapid, sensitive and was validated for streptomycin in accordance with SANCO/3131/2007. After extraction with phosphate buffer and a pH change, the clean-up was performed by the way of SPE with polymeric phase. The LC-MS/MS analysis was carried out using a HILIC column for the separation of the analytes and a triple quadrupole mass spectrometer in positive ESI mode to measure the transitions of the substances in MRM mode. For the quantification of streptomycin a matrix calibration curve in the linear range of 1.0-20 microg kg(-1) and the internal standard dihydrostreptomycin (10 microg kg(-1)) were used. The calculated validation parameters like the recovery (101-105%) for 2, 5, 10 and 20 microg kg(-1) and the relative standard deviation (RSD, 4.1-11.4%) of the 6 replicates fulfil the requirements of SANCO/3131/2007. The LOQ was determined as 2 microg kg(-1).

Array CGH in molecular diagnosis of mental retardation - A study of 150 Finnish patients.

We report on the results of an array comparative genomic hybridization (array CGH) study of 150 karyotypically normal Finnish patients with idiopathic mental retardation and/or dysmorphic features and/or malformations. Using high-resolution microarray analysis, we sought to identify clinically relevant microdeletions and microduplications in these patients. The results were confirmed using other methods and compared with findings reported in recent publications and internet databases. Small aberrations of potential clinical significance were found in 28 (18.6%) of the 150 patients. Eight of the identified aberrations are known to cause syndromes, 4 affected the X chromosome in males, 4 were familial, and 13 have yet to be associated with a phenotype. This study demonstrates the benefits of array CGH in clinical diagnostics of developmental disorders. Further, our findings give evidence of new syndromes.

Directed overexpression of insulin in Leydig cells causes a progressive loss of germ cells.

The primary goal of this study was to determine the 5'region of the Insl3 gene that specifically targets the expression of human insulin to Leydig cells, and to explore whether the testicular proinsulin is efficiently processed to insulin that is able to rescue the diabetes in different mouse models of diabetes. We show here that the sequence between nucleotides -690 and +4 of mouse Insl3 promoter is sufficient to direct the Leydig cell-specific expression of the human insulin transgene (Insl3-hIns). We also found that the 3'untranslated region (3'UTR) of Insl3 was effective in enhancing transgene expression of the insulin in vivo. Expression analysis revealed that the temporal expression pattern of the hIns transgene in Leydig cells of transgenic testes is roughly the same as that of the endogenous Insl3. Despite the Leydig cells translate human proinsulin and secrete a significant level of free C-peptide into the serum, the Leydig cell-derived insulin is not able to overcome the diabetes in different mouse models of diabetes, suggesting a lack of glucose sensing mechanisms in the Leydig cells. A consequence of overexpression of the human proinsulin in Leydig cells was the decrease of fertility of transgenic males at older ages. Germ cells in transgenic males were able to initiate and complete spermatogenesis. However, there was a progressive and age-dependent degeneration of the germ cells that lead to male infertility with increasing age.

TSPY expression is variably altered in transgenic mice with testicular feminization.

TSPY (testis-specific protein, Y-encoded) genes are expressed in premeiotic germ cells and round spermatids. The topology and timing of TSPY expression, and also its homology to members of the TTSN-family, suggest that TSPY is a proliferation factor for germ cells. There is also evidence for a role of TSPY in the aetiology of testis cancer. TSPY is a candidate for GBY, the elusive gonadoblastoma locus on the human Y chromosome, which is thought to predispose dysgenetic gonads of 46, XY sex-reversed females to develop gonadoblastoma. We have previously generated a TSPY transgenic mouse line (Tg(TSPY)9Jshm) that carries approximately 50 copies of the human TSPY gene on the mouse Y chromosome. In order to elucidate TSPY expression under complete androgen insensitivity and to investigate a possible role of TSPY in gonadal tumorigenesis, we have now generated sex-reversed TSPY transgenic Ar(Tfm) mice hemizygous for the X-linked testicular feminization mutation (Ar(Tfm)). We can show that the TSPY transcript is aberrantly spliced in the testes of TSPY-Ar(Tfm) mice, and that TSPY expression is upregulated by androgen insensitivity in some but not all animals. TSPY transgenic mice showed significantly increased testes weights. In one TSPY transgenic Ar(Tfm) animal, spermatogenesis proceeded beyond meiotic prophase. No tumors of germ cell origin were found in the testes of TSPY-Ar(Tfm) mice. Five out of 46 TSPY transgenic Ar(Tfm) mice, and 3 out of 31 age-related NMRI-Ar(Tfm) controls developed Leydig cell tumors, whereas none of the age-matched Ar(Tfm) mice (n=44) on a wild type background were affected by Leydig cell tumorigenesis.

Mutations in the cyclin family member FAM58A cause an X-linked dominant disorder characterized by syndactyly, telecanthus and anogenital and renal malformations.

We identified four girls with a consistent constellation of facial dysmorphism and malformations previously reported in a single mother-daughter pair. Toe syndactyly, telecanthus and anogenital and renal malformations were present in all affected individuals; thus, we propose the name 'STAR syndrome' for this disorder. Using array CGH, qPCR and sequence analysis, we found causative mutations in FAM58A on Xq28 in all affected individuals, suggesting an X-linked dominant inheritance pattern for this recognizable syndrome.

Multigene deletions on chromosome 20q13.13-q13.2 including SALL4 result in an expanded phenotype of Okihiro syndrome plus developmental delay.

Okihiro syndrome results from truncating mutations in the SALL4 locus on the chromosome 20q13.13-q13.2. Deletions of the whole SALL4 coding region as well as single exon deletions are also a common cause of Okihiro syndrome and indicate haploinsufficiency as the disease causing mechanism. The phenotypes caused by SALL4 deletions are not different from those caused by point mutations. No multigene deletion including SALL4 has been documented to date. Here we report the detection and molecular characterization of four novel, overlapping microdeletions, all spanning SALL4 and flanking genes, in four unrelated cases with features of Okihiro syndrome and variable degrees of psychomotor delay. All deletions were first identified and mapped by quantitative Real Time PCR. Subsequently, three of four deletions were mapped in further detail by high-resolution array CGH (244k oligo-arrays). All cases had larger deletions of varying size (1.76-1.78 Mb, 2.01-2.05 Mb, 2.16-2.17 Mb, and 1.3-2.8 Mb, respectively), which included SALL4 plus 3 to 7 additional functional genes. While three cases with largely overlapping deletions are mildly developmentally delayed, the only patient with a more centromeric deletion is clearly mentally retarded. In this patient, four genes (MOCS3, DPM1, ADNP, BCAS4) are deleted, which were not affected in the other three cases, suggesting that the deletion of one or more of these genes contributes to the mental retardation. Since two of the four cases presented with choanal atresia, large deletions including SALL4 should be considered in the differential diagnosis of children with suspected CHARGE syndrome but without detectable CHD7 mutations.

Partial trisomy of distal 19q detected by quantitative real-time PCR and FISH in a girl with mild facial dysmorphism, hypotonia and developmental delay.

We report on a 2 7/12-year-old girl who was referred to us because of psychomotor developmental delay. She is the second child of healthy, non-consanguineous parents. Pregnancy and birth were uneventful. Milestones of motor development were delayed: grasping at 6 months, sitting without support at 16 months, crawling at 16 months and walking at 2 4/12 years of age. She spoke about five words and followed simple instructions. Banding cytogenetics revealed a numerically and structurally normal female karyotype of 46,XX. By quantitative real-time PCR analysis of all subtelomeric regions, a partial trisomy of the subtelomeric region of 19q could be detected. This result was confirmed by FISH-analysis with a subtelomeric probe for 19q. The additional material of chromosome 19q was localized on chromosome 6q. However, a deletion of the subtelomeric region of 6q could not be detected with a subtelomeric FISH probe for 6q. Conventional cytogenetic analysis as well as FISH with subtelomeric probes for 19q and 6q showed normal results in the parents. The detected chromosomal aberration probably occurred de novo. The clinical features are very likely to be caused solely by the partial trisomy 19q.

Asthenoteratozoospermia in mice lacking testis expressed gene 18 (Tex18).

Testis expressed gene 18 (Tex18) is a small gene with one exon of 240 bp, which is specifically expressed in male germ cells. The gene encodes for a protein of 80 amino acids with unknown domain. To investigate the function of (Tex18) gene, we generated mice with targeted disruption of the (Tex18) gene by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fertile, while they are subfertile on the 129/Sv background, although mating is normal. We showed that Tex18(-/-) males are subfertile because of abnormal sperm morphology and reduced motility, which is called asthenoteratozoospermia, suggesting that (Tex18) affects sperm characteristics. Maturation of spermatids is unsynchronized and partially impaired in seminiferous tubules of Tex18(-/-) mice. Electron microscopical examination demonstrated abnormal structures of sperm head. In vivo experiments with sperm of Tex18(-/-) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility, as determined by the computer-assisted semen analysis system, is significantly affected, compared to wild-type spermatozoa. Generation of transgenic mice containing Tex18-EGFP fusion construct revealed a high transcriptional activity of (Tex18) during spermiogenesis, a process with morphological changes of haploid germ cells and development to mature spermatozoa. These results indicate that (Tex18) is expressed predominantly during spermatid differentiation and subfertility of the male Tex18(-/-) mice on the 129/Sv background is due to the differentiation arrest, abnormal sperm morphology and reduced sperm motility.

Premature translation of transition protein 2 mRNA causes sperm abnormalities and male infertility.

During mammalian spermiogenesis somatic histones are replaced at first by transition proteins, which are in turn replaced by the protamines, forming the sperm nucleoprotamines. It is believed that transition protein 2 (Tnp2) is necessary for maintaining the normal processing of protamines and, consequently, the completion of chromatin condensation. The transition protein mRNAs are stored in translationally inert messenger ribonucleoprotein particles for up to 7 days until translational activation in elongated spermatids. Substantial evidence suggests an involvement of 3'untranslated region (UTR) in the translational regulation of the Tnp2 mRNAs. In order to determine the role of Tnp2 3'UTR in translational regulation and to study whether the translational repression of Tnp2 mRNA is necessary for normal spermatid differentiation in mice, we generated transgenic mice that carry a Tnp2-hGH transgene. In this transgene, 3'UTR of Tnp2 gene was replaced by 3' 3'UTR of human growth hormone gene. In these transgenic animals, transcription and translation of Tnp2 occur simultaneously in round spermatids which is an evidence for involvement of Tnp2 3'UTR in its translation repression. Premature translation of Tnp2 mRNA caused abnormal head morphogenesis, reduced sperm motility and male infertility. These results show clearly that a strict temporal and stage-specific Tnp2 translation is necessary for the correct differentiation of round spermatids into mature spermatozoa and for male fertility.

Disruption of PLC-beta 1-mediated signal transduction in mutant mice causes age-dependent hippocampal mossy fiber sprouting and neurodegeneration.

Aberrant reorganization of hippocampal mossy fibers occurs in human temporal lobe epilepsy and rodent epilepsy models. We generated a mouse model showing massive late-onset aberrant mossy fiber sprouting in the adult hippocampus. The mutation in this mouse model derives from an intronic insertion of transgene DNA in the mouse PLC-beta1 gene (PLC-beta 1(-/-)(TC) mutation) leading to a splice mutation of the PLC-beta 1 gene and a complete loss of downstream PLC-beta 1 expression. PLC-beta 1(-/-)(TC) mutants develop a loss of NMDA-receptors in the stratum oriens of region CA1, apoptotic neuronal death, and reduced hippocampal PKC activity. The phenotype of these mice further consists of a late-onset epileptiform hyperexcitability, behavioral modifications in a radial maze and in an open field, female nurturing defect, and male infertility. In the present study, we provide evidence that the arising of the behavioral phenotype in PLC-beta 1(-/-)(TC) mice correlates in time with the development of the aberrant mossy fiber projections and that the disruption of the PLC-beta 1-mediated signal transduction pathway may lead to a functional cholinergic denervation, which could cause hippocampal remodeling and, in consequence, epileptiform hyperexcitability.