Jörg Schröder

SMALL DELETION/INSERTION MUTATIONS WITHIN POLY-A RUNS OF THE FACTOR VIII GENE MITIGATE THE SEVERE HAEMOPHILIA A PHENOTYPE

Recently Young et al. [1] reported a haemophilia A family in whom a single base deletion within an A nm of exon 14 of the factor VIII (FVIII) gene did not lead to the severe phenotype that is typically expected from frame shift mutations. We wish to point out that mitigation of the severe haemophilia A phenotype in patients with small deletion/insertion mutations of exon 14 may not be rare.

Targeted next-generation sequencing of deafness genes in hearing-impaired individuals uncovers informative mutations

Purpose:Targeted next-generation sequencing provides a remarkable opportunity to identify variants in known disease genes, particularly in extremely heterogeneous disorders such as nonsyndromic hearing loss. The present study attempts to shed light on the complexity of hearing impairment.Methods:Using one of two next-generation sequencing panels containing either 80 or 129 deafness genes, we screened 30 individuals with nonsyndromic hearing loss (from 23 unrelated families) and analyzed 9 normal-hearing controls.Results:Overall, we found an average of 3.7 variants (in 80 genes) with deleterious prediction outcome, including a number of novel variants, in individuals with nonsyndromic hearing loss and 1.4 in controls. By next-generation sequencing alone, 12 of 23 (52%) probands were diagnosed with monogenic forms of nonsyndromic hearing loss; one individual displayed a DNA sequence mutation together with a microdeletion. Two (9%) probands have Usher syndrome. In the undiagnosed individuals (10/23; 43%) we detected a significant enrichment of potentially pathogenic variants as compared to controls.Conclusion:Next-generation sequencing combined with microarrays provides the diagnosis for approximately half of the GJB2 mutation–negative individuals. Usher syndrome was found to be more frequent in the study cohort than anticipated. The conditions in a proportion of individuals with nonsyndromic hearing loss, particularly in the undiagnosed group, may have been caused or modified by an accumulation of unfavorable variants across multiple genes.Genet Med 16 12, 945–953.

Heterozygous large deletions of Factor 8 gene in females identified by multiplex PCR‐LC

Summary.  Haemophilia A is the most common X‐linked recessive bleeding disorder. In 5% of severely affected patients the mutations responsible for the disease are large deletions encompassing from one exon to the complete Factor 8 (F8) gene. Large deletions in a male haemophilic patient are easily detected by the absence of the corresponding PCR product. However, in female carriers, identification of the various heterozygous large deletions is difficult representing a major limitation to accurate carrier diagnosis. The deletion is masked by the presence of the second allele that serves as template for the PCR reaction. Quantitative PCR can differentiate between the presence of one or two alleles. Here we report an assay based on multiplex amplification of several exons of the F8 gene of various length and subsequent quantitative evaluation of the amplicons by liquid chromatogphy (LC). Using this approach we achieved an accurate classification of 16 female carriers and eight non‐carriers for deletions in the F8 gene in 19 investigated families. One mother and one grandmother were classified as non‐carriers, underlining the high de novo mutation rate of large deletions in female germ cells. The large deletions in three families were confirmed by fluorescent in situ hybridization. In conclusion, the multiplex PCR‐LC technique represents a rapid, simple and reliable method for detection of heterozygous large deletions in female carriers.

Letters to the Editor: Genetic background and inhibitors in previously untreated or minimally treated young patients with severe haemophilia A treated with sucrose-formulated recombinant factor VIII

Letters to the Editor: Genetic background and inhibitors in previously untreated or minimally treated young patients with severe haemophilia A treated with sucrose-formulated recombinant factor VIII -

A Novel de novo Mutation in CEACAM16 Associated with Postlingual Hearing Impairment

Mutations in CEACAM16 cause autosomal dominant nonsyndromic hearing loss (DFNA4B). So far, 2 families have been reported with segregating missense mutations, both in the immunoglobulin constant domain A of the CEACAM16 protein. In this study, we used the TruSight One panel to investigate a parent-child trio without familial history of hearing loss and one affected child. When filtering for recessive inheritance and de novo events, we discovered a de novo CEACAM16 mutation (c.1094T>G, p.Leu365Arg) as the sole likely pathogenic variant. The de novo mutation was confirmed by Sanger sequencing and STR analysis. The probands hearing loss closely matches the described onset and severity for DFNA4B. We present the third CEACAM16 variant and the first de novo mutation in CEACAM16. This de novo mutation is robustly described as a pathogenic mutation according to in silico mutation prediction tools and affects a highly conserved amino acid in the most strongly conserved CEACAM16 N2 domain. Our strategy of screening family trios enhances de novo mutation discovery and the exclusion of other variants of potential interest through pedigree filtering.

Investigation of Underlying Reasons of Factor VIII Deficiency in Hemophilia A Patients with Undetectable Mutations in the F8 Gene

Hemophilia A (HA) is an X-linked bleeding disorder caused by heterogeneous mutations in the coagulation factor VIII (F8) gene [4]. Despite applying sensitive methods for mutation detection, and after excluding the inversions mutations a causative mutation is not identified in F8 gene in about 2.5% of severe HA patients (53 patients out of 2350 German patients) (unpublished data). Analysis of mRNA from a small group of such (German) patients has excluded mutations deep in the introns that may affect normal splicing or mechanisms causing some unknown rearrangements of the F8 gene as the cause of HA [1]. Among this group, in one patient no F8 mRNA was detected [2]. Using two common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother and his sister was not detected by reverse transcription PCR (RT-PCR) from total blood mRNA. These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA which points to a novel mechanism leading to HA. Recently we established an international multi-centre collaborative study to assemble a large collection of such families.

High mating success of low rank males in Limia perugiae (Pisces: Poeciliidae) as determined by DNA fingerprinting

Hierarchical structures among male individuals in a population are frequently reflected in differences in aggressive and reproductive behaviour and access to the females. In general social dominance requires large investments which in turn may have to be compensated for by high reproductive success. However, this hypothesis has so far only been sufficiently tested in small mating groups due to the difficulties of determining paternity by classical methods using non-molecular markers. DNA fingerprinting overcomes these problems offering the possibility to determine genetic relationships and mating patterns within larger groups. Using this approach we have recently shown (Schartl et al., 1993) that in the poeciliid fish Limia perugiae in small mating groups the dominant male has 100% mating success, while in larger groups its contribution to the offspring unexpectedly drops to zero. The reproductive failure under such social conditions is explained by the inability of the α-male to protect all the females simultaneously against mating attempts of his numerous subordinate competitors.

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation-negative early-onset and high-risk breast cancer patients: Epigenetic abnormalities of tumor suppressor genes in breast cancer patients

To evaluate the role of constitutive epigenetic changes in normal body cells of BRCA1/BRCA2‐mutation negative patients, we have developed a deep bisulfite sequencing assay targeting the promoter regions of 8 tumor suppressor (TS) genes (BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, RB1) and the estrogene receptor gene (ESR1), which plays a role in tumor progression. We analyzed blood samples of two breast cancer (BC) cohorts with early onset (EO) and high risk (HR) for a heterozygous mutation, respectively, along with age‐matched controls. Methylation analysis of up to 50,000 individual DNA molecules per gene and sample allowed quantification of epimutations (alleles with >50% methylated CpGs), which are associated with epigenetic silencing. Compared to ESR1, which is representative for an average promoter, TS genes were characterized by a very low (< 1%) average methylation level and a very low mean epimutation rate (EMR; < 0.0001% to 0.1%). With exception of BRCA1, which showed an increased EMR in BC (0.31% vs. 0.06%), there was no significant difference between patients and controls. One of 36 HR BC patients exhibited a dramatically increased EMR (14.7%) in BRCA1, consistent with a disease‐causing epimutation. Approximately one third (15 of 44) EO BC patients exhibited increased rates of single CpG methylation errors in multiple TS genes. Both EO and HR BC patients exhibited global underexpression of blood TS genes. We propose that epigenetic abnormalities in normal body cells are indicative of disturbed mechanisms for maintaining low methylation and appropriate expression levels and may be associated with an increased BC risk.

Hereditary hearing loss SNP-microarray pilot study

ObjectivesDespite recent advancements in diagnostic tools, the genomic landscape of hereditary hearing loss remains largely uncharacterized. One strategy to understand genome-wide aberrations includes the analysis of copy number variation that can be mapped using SNP-microarray technology. A growing collection of literature has begun to uncover the importance of copy number variation in hereditary hearing loss. This pilot study underpins a larger effort that involves the stage-wise analysis of hearing loss patients, many of whom have advanced to high-throughput sequencing analysis.Data descriptionOur data originate from the Infinium HumanOmni1-Quad v1.0 SNP-microarrays (Illumina) that provide useful markers for genome-wide association studies and copy number variation analysis. This dataset comprises a cohort of 108 individuals (99 with hearing loss, 9 normal hearing family members) for the purpose of understanding the genetic contribution of copy number variations to hereditary hearing loss. These anonymized SNP-microarray data have been uploaded to the NCBI Gene Expression Omnibus and are intended to benefit other investigators interested in aggregating platform-matched array patient datasets or as part of a supporting reference tool for other laboratories to better understand recurring copy number variations in other genetic disorders.

Letters to the Editor: Haemophilia A in a female caused by coincidence of a Swyer syndrome and a missense mutation in factor VIII gene

Letters to the Editor: Haemophilia A in a female caused by coincidence of a Swyer syndrome and a missense mutation in factor VIII gene -