CRISPR-Cas9-Mediated Cytosine Base Editing Screen for the Functional Assessment of CALR Intron Variants in Japanese Encephalitis Virus Replication.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the CALR gene.

The complete chloroplast genome sequences of eight Orostachys species: Comparative analysis and assessment of phylogenetic relationships.

We analyzed the complete chloroplast genomes of eight Orostachys species and compared the sequences to those of published chloroplast genomes of the congeneric and closely related genera, Meterostachys and Hylotelephium. The total chloroplast genome length of thirteen species, including the eight species analyzed in this study and the five species analyzed in previous studies, ranged from 149,860 (M. sikokianus) to 151,707 bp (H. verticillatum). The overall GC contents of the genomes were almost identical (37.6 to 37.8%). The thirteen chloroplast genomes each contained 113 unique genes comprising 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Among the annotated genes, sixteen genes contained one or two introns. Although the genome structures of all Orostachys and Hylotelephium species were identical, Meterostachys differed in structure due to a relatively large gene block (trnS-GCU-trnS-GGA) inversion. The nucleotide diversity among the subsect. Orostachys chloroplast genomes was extremely low in all regions, and among the subsect. Appendiculatae, genus Orostachys, and all thirteen chloroplast genomes showed high values of Pi (>0.03) in one, five, or three regions. The phylogenetic analysis showed that Orostachys formed polyphyly, and subsect. Orostachys and Appendiculatae were clustered with Hylotelephium and Meterostachys, respectively, supporting the conclusion that each subsection should be considered as an independent genus. Furthermore, the data supported the taxonomic position of O. margaritifolia and O. iwarenge f. magnus, which were treated as synonyms for O. iwarenge in a previous study, as independent taxa. Our results suggested that O. ramosa and O. japonica f. polycephala were individual variations of O. malacophylla and O. japonica, respectively. The exact taxonomic position of O. latielliptica and the phylogenetic relationship among the three species, O. chongsunensis, O. malacophylla and O. ramosa, should be a topic of future study.

Assessment of the association between NUS1 variants and essential tremor.

BACKGROUND: A recent study on early onset Parkinson's disease (PD) revealed that NUS1 is a risk gene for PD. Clinically, essential tremor (ET) is closely related to PD. In this study, we aimed to detect NUS1 variants and assess the effect of those variants on patients with ET. METHODS: The 5 coding regions and the exon-intron boundaries of NUS1 were directly sequenced in 395 patients with ET and an equal number of healthy controls, matched for age and sex. The function of variants was assessed by pathogenic predictive software programs. Genetic analysis of variants was used to evaluate susceptibility to ET. RESULTS: A total of 6 exonic variants were identified, including 3 synonymous and 3 missense variants. The non-synonymous variants were predicted to be tolerable. No variants had significant association with ET (none of the p-values were less than 0.05, using Fisher's exact test). CONCLUSION: Our study suggested that NUS1 variants may not contribute to the risk of ET.

Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies.

Elucidating the functional consequence of molecular defects underlying genetic diseases enables appropriate design of therapeutic options. Treatment of cystic fibrosis (CF) is an exemplar of this paradigm as the development of CFTR modulator therapies has allowed for targeted and effective treatment of individuals harboring specific genetic variants. However, the mechanism of these drugs limits effectiveness to particular classes of variants that allow production of CFTR protein. Thus, assessment of the molecular mechanism of individual variants is imperative for proper assignment of these precision therapies. This is particularly important when considering variants that affect pre-mRNA splicing, thus limiting success of the existing protein-targeted therapies. Variants affecting splicing can occur throughout exons and introns and the complexity of the process of splicing lends itself to a variety of outcomes, both at the RNA and protein levels, further complicating assessment of disease liability and modulator response. To investigate the scope of this challenge, we evaluated splicing and downstream effects of 52 naturally occurring CFTR variants (exonic = 15, intronic = 37). Expression of constructs containing select CFTR intronic sequences and complete CFTR exonic sequences in cell line models allowed for assessment of RNA and protein-level effects on an allele by allele basis. Characterization of primary nasal epithelial cells obtained from individuals harboring splice variants corroborated in vitro data. Notably, we identified exonic variants that result in complete missplicing and thus a lack of modulator response (e.g. c.2908G>A, c.523A>G), as well as intronic variants that respond to modulators due to the presence of residual normally spliced transcript (e.g. c.4242+2T>C, c.3717+40A>G). Overall, our data reveals diverse molecular outcomes amongst both exonic and intronic variants emphasizing the need to delineate RNA, protein, and functional effects of each variant in order to accurately assign precision therapies.

SimSpliceEvol: alternative splicing-aware simulation of biological sequence evolution.

BACKGROUND: It is now well established that eukaryotic coding genes have the ability to produce more than one type of transcript thanks to the mechanisms of alternative splicing and alternative transcription. Because of the lack of gold standard real data on alternative splicing, simulated data constitute a good option for evaluating the accuracy and the efficiency of methods developed for splice-aware sequence analysis. However, existing sequence evolution simulation methods do not model alternative splicing, and so they can not be used to test spliced sequence analysis methods. RESULTS: We propose a new method called SimSpliceEvol for simulating the evolution of sets of alternative transcripts along the branches of an input gene tree. In addition to traditional sequence evolution events, the simulation also includes gene exon-intron structure evolution events and alternative splicing events that modify the sets of transcripts produced from genes. SimSpliceEvol was implemented in Python. The source code is freely available at https://github.com/UdeS-CoBIUS/SimSpliceEvol. CONCLUSIONS: Data generated using SimSpliceEvol are useful for testing spliced RNA sequence analysis methods such as methods for spliced alignment of cDNA and genomic sequences, multiple cDNA alignment, orthologous exons identification, splicing orthology inference, transcript phylogeny inference, which requires to know the real evolutionary relationships between the sequences.

Comparative assessment of chloroplast transcriptional responses highlights conserved and unique patterns across Triticeae members under salt stress.

Chloroplast functional genomics, in particular understanding the chloroplast transcriptional response is of immense importance mainly due to its role in oxygenic photosynthesis. As a photosynthetic unit, its efficiency and transcriptional activity is directly regulated by reactive oxygen species during abiotic and biotic stress and subsequently affects carbon assimilation, and plant biomass. In crops, understanding photosynthesis is crucial for crop domestication by identifying the traits that could be exploited for crop improvement. Transcriptionally and translationally active chloroplast plays a key role by regulating the PSI and PSII photo-reaction centres, which ubiquitously affects the light harvesting. Using a comparative transcriptomics mapping approach, we identified differential regulation of key chloroplast genes during salt stress across Triticeae members with potential genes involved in photosynthesis and electron transport system such as CytB6f. Apart from differentially regulated genes involved in PSI and PSII, we found widespread evidence of intron splicing events, specifically uniquely spliced petB and petD in Triticum aestivum and high proportion of RNA editing in ndh genes across the Triticeae members during salt stress. We also highlight the role and differential regulation of ATP synthase as member of CF0CF1 and also revealed the effect of salt stress on the water-splitting complex under salt stress. It is worthwhile to mention that the observed conserved down-regulation of psbJ across the Triticeae is limiting the assembly of water-splitting complexes and thus making the BEP clade Triticeae members more vulnerable to high light during the salt stress. Comparative understanding of the chloroplast transcriptional dynamics and photosynthetic regulation will improve the approaches for improved crop domestication.

Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition.

PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants. METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay. RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities. CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

Genome-wide analysis of basic/helix-loop-helix gene family in peanut and assessment of its roles in pod development.

The basic/helix-loop-helix (bHLH) proteins constitute a superfamily of transcription factors that are known to play a range of regulatory roles in eukaryotes. Over the past few decades, many bHLH family genes have been well-characterized in model plants, such as Arabidopsis, rice and tomato. However, the bHLH protein family in peanuts has not yet been systematically identified and characterized. Here, 132 and 129 bHLH proteins were identified from two wild ancestral diploid subgenomes of cultivated tetraploid peanuts, Arachis duranensis (AA) and Arachis ipaensis (BB), respectively. Phylogenetic analysis indicated that these bHLHs could be classified into 19 subfamilies. Distribution mapping results showed that peanut bHLH genes were randomly and unevenly distributed within the 10 AA chromosomes and 10 BB chromosomes. In addition, 120 bHLH gene pairs between the AA-subgenome and BB-subgenome were found to be orthologous and 101 of these pairs were highly syntenic in AA and BB chromosomes. Furthermore, we confirmed that 184 bHLH genes expressed in different tissues, 22 of which exhibited tissue-specific expression. Meanwhile, we identified 61 bHLH genes that may be potentially involved in peanut-specific subterranean. Our comprehensive genomic analysis provides a foundation for future functional dissection and understanding of the regulatory mechanisms of bHLH transcription factors in peanuts.

A fast and cost-effective molecular diagnostic tool for genetic diseases involved in sudden cardiac death.

BACKGROUND: Cardiomyopathies and arrhythmia syndromes are common genetic cardiac diseases that account for a significant number of sudden cardiac death (SCD) cases. METHODS: NGS workflow based on a panel of 95 genes was developed on Illumina NextSeq500™ sequencer for sequencing prevalent SCD-causing genes. A cohort of 90 patients (56 genotype-positive, 27 genotype-negative and 7 new cases) was screened to evaluate this strategy in terms of sensitivity, specificity, practicability and cost. In silico analysis were performed using a pipeline based on NextGENe® software and a personalized Sophia Genetics pipeline. RESULTS: Using our panel custom, 100% of targeted sequences were efficiently covered and all previously identified genetic variants were readily detected. Applied to 27 genotype-negative patients, this molecular strategy allowed the identification of pathogenic or likely pathogenic variants into 12 cases. It confirmed the involvement of HCN4 mutations in the combined bradycardia­myocardial non-compaction phenotype, and also suggested, for the first time, the involvement of PKP2, usually associated with arrhythmogenic right ventricular dysplasia, in ventricular non-compaction. CONCLUSION: This NGS approach is a fast, cheap, sensitive and high-throughput mutation detection method that is ready to be deployed in clinical laboratories and would provide new insights on physiopathology of SCD, more particularly of cardiomyopathies and arrhythmia syndromes.

Comprehensive assessment of genetic variants within TCF4 in Fuchs' endothelial corneal dystrophy.

PURPOSE: The single nucleotide variant (SNV), rs613872, in the transcription factor 4 (TCF4) gene was previously found to be strongly associated (P = 6 × 10(-26)) with Fuchs' endothelial corneal dystrophy (FECD). Subsequently, an intronic expansion of the repeating trinucleotides, TGC, was found to be even more predictive of disease. We performed comprehensive sequencing of the TCF4 gene region in order to identify the best marker for FECD within TCF4 and to identify other novel variants that may be associated with FECD. METHODS: Leukocyte DNA was isolated from 68 subjects with FECD and 16 unaffected individuals. A custom capture panel was used to isolate the region surrounding the two previously validated markers of FECD. Sequencing of the TCF4 coding region, introns and flanking sequence, spanning 465 kb was performed at >1000× average coverage using the Illumina HiSequation 2000. RESULTS: TGC expansion (>50 repeats) was present in 46 (68%) FECD-affected subjects and one (6%) normal subject. A total of 1866 variants, including 1540 SNVs, were identified. Only two previously reported SNVs resided in the TCF4 coding region, neither of which segregated with disease. No variant, including TGC expansion, correlated perfectly with disease status. Trinucleotide repeat expansion was a better predictor of disease than any other variant. CONCLUSIONS: Complete sequencing of the TCF4 genomic region revealed no single causative variant for FECD. The intronic trinucleotide repeat expansion within TCF4 continues to be more strongly associated with FECD than any other genetic variant.

Genetic variations of b-MYH7 in Venezuelan patients with hypertrophic cardiomyopathy / Variaciones genéticas en el gen MYH7 en pacientes venezolanos con miocardiopatía hipertrófica

Hypertrophic cardiomyopathy (HCM) is a cardiac disease, characterized by marked hypertrophy and genetic variability. HCM has been associated with sarcomere protein mutations, being cardiac b-myosin (coded by the MYH7 gene) and myosin binding protein C (coded by the MYBPC3 gene) the most frequently affected proteins. As in Venezuela only the clinical analysis are performed in HCM patients, we decided to search for genetic variations in the MYH7 gene. Coding regions, including the junction exon-intron of the MYH7 gene, were studied in 58 HCM patients, whose samples were collected at the ASCARDIO Hospital (Barquisimeto, Lara state, Venezuela) and 106 control subjects from the ASCARDIO Hospital and the IVIC (Barquisimeto Lara state and Miranda, Venezuela, respectively). The blood samples were analyzed by genomic DNA isolation, followed by polymerase chain reaction and sequence analysis. The screening of the MYH7 gene revealed eight already reported polymorphic variants, as well as two intronic variations in these HCM patients. Neither any missense mutations nor other pathological mutations in the MYH7 gene were found in the HCM patients.

La miocardiopatía hipertrófica (MH) es una enfermedad cardiaca primaria, caracterizada por una marcada hipertrofia y variabilidad genética. MH ha sido asociada con mutaciones en las proteínas del sarcómero, siendo la beta miosina cardiaca, codificada por el gen MYH7 y la proteína de unión a miosina C, codificada por el gen MYBPC3, las principalmente afectadas. En Venezuela únicamente se realiza el diagnóstico clínico de MH, por lo cual el objetivo principal de este trabajo fue realizar el análisis genético en los pacientes, iniciando con el gen MYH7. Para ello, se estudió la región codificante, incluyendo la región de unión exón-intron del gen MYH7 en 58 pacientes provenientes de ASCARDIO (Barquisimeto, estado Lara) y 106 controles provenientes de ASCARDIO e IVIC (estados Lara y Miranda, Venezuela). Se colectaron las muestras de sangre para el aislamiento del ADN genómico, se realizó la técnica de PCR, seguido del análisis de secuencias para la detección de mutaciones en pacientes y controles. Se encontraron 8 polimorfismos previamente reportados, y 2 variaciones intrónicas. No se encontraron mutaciones que involucraran un cambio de aminoácido en ninguno de los exones del gen MYH7 de la beta miosina cardiaca.

Statistical analysis of combinatorial transcriptional regulatory motifs in human intron-containing promoter sequences.

The combinatorial regulation is an important feature of transcriptional regulation of genes. Although a number of researches have been done, the general properties of combinatorial transcriptional regulatory elements, including their patterns and distributions, are still not well understood. Specially, the understanding for laws of transcriptional regulation involving introns is challenging. Here, we performed statistical analyses on potentially interacting motif pairs in human housekeeping genes and tissue-specific genes of some tissues. We first identified transcriptional regulatory motifs in promoter sequences, including the first introns, using a sequence-based approach which incorporates several different sequence backgrounds. And then we predicted the potentially interacting motif pairs using Hypergeometric distribution and Mann-Whitney U-test. The validation of the predicted interacting motif pairs has been carried out via analyses of orthologous conservation and enrichment. The distribution characteristics of the identified motif pairs were examined using a probability model. We found that interacting motif pairs have some significant preferences in location, distance and orientation in housekeeping and tissue-specific genes. The results suggest that the features of transcriptional regulation in housekeeping genes are different from the tissue-specific genes. Besides, location preferences of the motif pairs provide clues to the way of the introns participating in transcriptional regulation in different tissue-specific genes.

Association of a BMP5 microsatellite with knee osteoarthritis: case-control study.

INTRODUCTION: We aimed to explore the involvement of a multiallelic functional polymorphism in knee osteoarthritis (OA) susceptibility as a prototype of possible genetic factors escaping GWAS detection. METHODS: OA patients and controls from three European populations (Greece, Spain and the UK) adding up to 1003 patients (716 women, 287 men) that had undergone total knee joint replacement (TKR) due to severe primary OA and 1543 controls (758 women, 785 men) lacking clinical signs or symptoms of OA were genotyped for the D6S1276 microsatellite in intron 1 of BMP5. Genotype and mutiallelic trend tests were used to compare cases and controls. RESULTS: Significant association was found between the microsatellite and knee OA in women (P from 3.1 x10-4 to 4.1 x10-4 depending on the test), but not in men. Three of the alleles showed significant differences between patients and controls, one of them of increased risk and two of protection. The gender association and the allele direction of change were very concordant with those previously reported for hip OA. CONCLUSIONS: We have found association of knee OA in women with the D6S1276 functional microsatellite that modifies in cis the expression of BMP5 making this a sounder OA genetic factor and extending its involvement to other joints. This result also shows the interest of analysing other multiallelic polymorphisms.

Assessment of TP53 mutations in benign and malignant salivary gland neoplasms.

Despite advances in the understanding of the pathogenesis of salivary gland neoplasms (SGN), the molecular pathways associated with enhanced tumor growth and cell survival remain to be established. The aim of the present study was to investigate whether TP53 mutations are relevant to SGN pathogenesis and if they impact on p53 protein expression. The study included 18 benign and 18 malignant SGN samples. Two polymorphic microsatellite markers at the TP53 genetic locus were chosen to assess loss of heterozygosity (LOH) in the samples that had matched normal DNA. The TP53 exons 2-11 were amplified by PCR, and all of the products were sequenced. Reverse transcription-PCR of the TP53 open reading frame (ORF) was carried out in the samples that had fresh tissue available, and immunohistochemistry for the p53 protein was performed in all samples. TP53 LOH was only found in two pleomorphic adenomas. We found two missense mutations in exon 7 (one in a pleomorphic adenoma and the other in a polymorphous low grade adenocarcinoma), another in exon 8 (in a carcinoma ex pleomorphic adenoma) and a fourth missense mutation in exon 10 (in a mucoepidermoid carcinoma). In addition, a nonsense mutation was found in exon 8 of an adenoid cystic carcinoma. Several intronic and exonic SNPs were detected. Although almost all of the malignant samples were immunopositive for p53, approximately 37% of the benign samples were positive, including the sample harboring the missense mutation and one of the samples that showed LOH. The complete TP53 ORF could be amplified in all samples analyzed, including the IHC negative samples, the samples showing LOH and one sample displaying a missense mutation. In summary, our results show that TP53 mutations are not a frequent event in SGN and that p53 immunopositivity might not be associated with sequence mutations in SGN.

Assessment of epigenetic contributions to sexually-dimorphic Kiss1 expression in the anteroventral periventricular nucleus of mice.

The Kiss1 gene, which encodes kisspeptin and is critical for reproduction, is sexually differentiated in the hypothalamic anteroventral periventricular (AVPV)/rostral periventricular (PeN) nuclei. Specifically, female rodents have higher AVPV/PeN Kiss1 expression than males, but how this Kiss1 sex difference is induced in early development is poorly understood. Here, we explored the contribution of epigenetic mechanisms to the establishment of the AVPV/PeN Kiss1 sex difference, focusing on histone deacetylation and DNA methylation. First, we utilized postnatal pharmacological blockade of histone deacetylation and analyzed Kiss1 expression in the AVPV/PeN. Postnatal disruption of histone deacetylase modestly increased AVPV Kiss1 cell number in both sexes but did not alter the Kiss1 sex difference. Next, we assessed whether the level of CpG methylation, which can influence transcription factor binding and gene expression, in the murine Kiss1 gene differs between males and females. We found significant sex differences in methylation at several CpG sites in the putative promoter and first intron of the Kiss1 gene in the AVPV/PeN, but not in the arcuate (which lacks adult Kiss1 sex differences), suggesting that differential methylation of the Kiss1 gene may influence sexually-dimorphic Kiss1 expression in the AVPV/PeN. Transgenic impairment of methyl CpG-binding protein-2 function did not eliminate the Kiss1 sex difference, indicating that other methylation factors are involved. Interestingly, CpG methylation in the AVPV/PeN was lower in males than females, suggesting that transcriptional repressors may contribute to the AVPV/PeN Kiss1 sex difference, a possibility supported by in silico identification of putative repressor binding sites near some of the sexually-dimorphic CpG.

Influence of single nucleotide polymorphisms in factor VIII and von Willebrand factor genes on plasma factor VIII activity: the ARIC Study.

Factor VIII (FVIII) functions as a cofactor for factor IXa in the contact coagulation pathway and circulates in a protective complex with von Willebrand factor (VWF). Plasma FVIII activity is strongly influenced by environmental and genetic factors through VWF-dependent and -independent mechanisms. Single nucleotide polymorphisms (SNPs) of the coding and promoter sequence in the FVIII gene have been extensively studied for effects on FVIII synthesis, secretion, and activity, but impacts of non-disease-causing intronic SNPs remain largely unknown. We analyzed FVIII SNPs and FVIII activity in 10,434 healthy Americans of European (EA) or African (AA) descent in the Atherosclerosis Risk in Communities (ARIC) study. Among covariates, age, race, diabetes, and ABO contributed 2.2%, 3.5%, 4%, and 10.7% to FVIII intersubject variation, respectively. Four intronic FVIII SNPs associated with FVIII activity and 8 with FVIII-VWF ratio in a sex- and race-dependent manner. The FVIII haplotypes AT and GCTTTT also associated with FVIII activity. Seven VWF SNPs were associated with FVIII activity in EA subjects, but no FVIII SNPs were associated with VWF Ag. These data demonstrate that intronic SNPs could directly or indirectly influence intersubject variation of FVIII activity. Further investigation may reveal novel mechanisms of regulating FVIII expression and activity.

Population mutation scanning of human GHR by meltMADGE and identification of a paucimorphic variant.

Current studies of human genetic diversity are focused in two areas: first, detection of rare mutations in highly selected clinical cases; and second, in common single-nucleotide polymorphism (SNP) and haplotype effects in the general population. Less frequent SNPs and "paucimorphisms" remain underexplored, although lower frequency coding SNPs are more likely to have functional impact. We have developed a cost-efficient mutation scanning technology, meltMADGE, for population mutation scanning. Previous research in GHR has explored its role in extreme (-3 SD) growth retardation and, subsequently, "moderate" (-2 SD) growth retardation cases. Here, we describe meltMADGE assays for the entire coding region of GHR. As a first step we have established long polymerase chain reaction subbanks for GHR from 2423 unselected subjects and have applied meltMADGE scanning assays of exons 4 and 5 to these subbanks. A novel paucimorphism present at 439+30A>C (allele frequency: 0.0021) in intron 5 (location chr5:42,695,221 in GRCh37/hg19) was identified in 10 individuals, confirmed by sequencing and analysis made for major phenotypic effects. This approach is relevant to the deep sampling of populations for less frequent sequence diversity, some of which is expected to exert significant phenotypic effects.

[Inhibitor development after early high exposure and cerebral haemorrhage. Costs and factor demand for a successful immunotolerance induction therapy]. / Inhibitorentwicklung nach früher hoher Exposition und Hirnblutung. Kosten einer erfolgreichen Immuntoleranztherapie.

Severe haemophilia A was diagnosed postpartum in a newborn. The mother was known as a conductor (intron 22 inversion) and an uncle had a persistently high titer inhibitor after failed ITI. Due to a cephalhaematoma, a high-dose pdFVIII substitution was given within the first days after birth. At the age of six month a severe cerebral haemorrhage occurred, making a high-dose pdFVIII substitution and neurosurgical intervention necessary. Several days later a porth-a-cath-system was implanted. The development of a high titer inhibitor occured six days later, an ITI was started according to the Bonn Protocol. Initially rFVIIa was given in addition to the pdFVIII substitution. Seven days after the beginning of treatment the inhibitor was no longer detectable. At monthly intervals the FVIII dosage was reduced until the dosage complied with a prophylaxis in severe haemophilia A. The duration of the ITI was nine months. A total of 30 mg rFVIIa and 276000 IU pdFVIII were used; costs in total: 280173.60 Euro.