Advances in DNA sequencing technologies for high resolution HLA typing

This communication describes our experience in large-scale G group-level high resolution HLA typing using three different DNA sequencing platforms – ABI 3730 xl, Illumina MiSeq and PacBio RS II. Recent advances in DNA sequencing technologies, so-called next generation sequencing (NGS), have brought breakthroughs in deciphering the genetic information in all living species at a large scale and at an affordable level. The NGS DNA indexing system allows sequencing multiple genes for large number of individuals in a single run. Our laboratory has adopted and used these technologies for HLA molecular testing services. We found that each sequencing technology has its own strengths and weaknesses, and their sequencing performances complement each other. HLA genes are highly complex and genotyping them is quite challenging. Using these three sequencing platforms, we were able to meet all requirements for G group-level high resolution and high volume HLA typing.     

Shewanella algae in acute gastroenteritis

Shewanella algae is an emerging bacteria rarely implicated as a human pathogen. Previously reported cases of S. algae have mainly been associated with direct contact with seawater. Here we report the isolation of S. algae as the sole etiological agent from a patient suffering from acute gastroenteritis with bloody diarrhoea. The bacterium was identified by automated identification system and 16S rRNA gene sequence analysis. Our report highlights the importance of looking for the relatively rare aetiological agents in clinical samples that does not yield common pathogens. It also underscores the usefulness of automated systems in identification of rare pathogens.     

microRNA profiling in three main stages during porcine spermatogenesis

BackgroundSpermatogenesis is an intricate biological event wherein an undifferentiated spermatogonium develops into mature sperms. MicroRNAs are a type of single strand small non-coding RNA molecule and are implicated in the regulation of many crucial pathways during cell proliferation, apoptosis, and differentiation.MethodHere, we present a comprehensive comparison of miRNA expression profiling in three main stages during porcine spermatogenesis using high-throughput sequencing.ResultsWe built three small RNA libraries for the testis, the epididymis and the ejaculated sperm from a Landrace boar, and in total obtained 3821 precursor hairpins encoding for 4761 mature miRNAs, of which 23 are miRNA*. Notably, 940 precursor miRNAs produced both the 5’- and 3’- strands as sister pairs, indicating the distinctive expression patterns of germ cell miRNAs. Additionally, 418 out of 710 co-expressed miRNAs were identified as being differentially expressed between libraries (P < 0.001). Apart from the sexual specific X chromosome, many miRNAs were found to be located on chromosome 12, which may play potential roles in spermatogenesis according to the result of synteny analysis with human and mouse. The Gene Ontology and KEGG pathway analysis revealed that the target genes of co-expressed miRNAs were highly involved in the cell cycle process, metal ion binding, modification of plasma membrane, and the p53 signal pathway.

Electronic supplementary material

The online version of this article (doi:10.1007/s10815-014-0406-x) contains supplementary material, which is available to authorized users.     

Genotype/phenotype analysis in Chinese laminin‐α2 deficient congenital muscular dystrophy patients

Laminin‐α2 deficient congenital muscular dystrophy (CMD) is an autosomal recessive disorder characterized by severe muscular dystrophy, which is typically associated with abnormal white matter. In this study, we assessed 43 CMD patients with typical white matter abnormality and laminin‐α2 deficiency (complete or partial) diagnosed by immunohistochemistry to determine the clinical and molecular genetic characteristics of laminin‐α2 deficient CMD. LAMA2 gene mutation analysis was performed by direct sequencing of genomic DNAs. Exonic deletion or duplication was identified by multiplex ligation‐dependent probe amplification (MLPA) and verified by high‐density oligonucleotide‐based CGH microarrays. Gene mutation analysis revealed 86 LAMA2 mutations (100%); 15 known and 37 novel. Among these mutations, 73.9% were nonsense, splice‐site or frameshift and 18.8% were deletions of one or more exons. Genetic characterization of affected families will be valuable in prenatal diagnosis of CMD in the Chinese population.     

Use of panel tests in place of single gene tests in the cancer genetics clinic

Improved technology has made it possible to test for mutations within multiple genes simultaneously. It is not clear when these gene ‘panels’ should be used in the hereditary cancer setting. These analyses were intended to guide panel testing criteria. Offering hereditary panel testing as a first and final, ‘single‐tier’, option was explored. A ‘two‐tiered’ approach, in which panel testing is offered reflexively following stricter criteria, was then applied to the same data. Within our cohort of 105 patients, the single‐tier approach was associated with a higher mutation detection rate (6.7% vs 3.8%) and variant of uncertain significance (VUS) rate (0.94 vs 0.23 average per person) compared to a two‐tiered approach. Of the VUSs also identified in other patients by another lab, 53% were classified differently between laboratories. Individuals reporting African American race had more VUSs compared to other ancestry groups (p = 0.001). The test cost for a single‐tier test was 21% more than a two‐tiered approach. Single‐tier panel testing was associated with higher mutation and VUS rates, and there is inconsistent classification of the VUS/low penetrant genes between laboratories.     

A mutation in the Z‐line Cypher/ZASP protein is associated with arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an important cause of malignant arrhythmia and sudden death particularly in young people. Although it is considered a desmosomal disease, mutations in non‐desmosomal genes have also been identified. We report on a family where a mutation in LDB3 is associated with this condition. The index case and first and second degree relatives underwent a complete clinical evaluation: physical examination, electrocardiography (ECG), signal‐averaged ECG, 2D echocardiogram, cardiac magnetic resonance and 24‐h monitoring. After ruling out mutations in the five desmosomal genes, genetic testing by means of Next Generation Sequencing was carried out on the proband. A heterozygous missense mutation in LDB3 c.1051A>G was identified. This result was confirmed by subsequent Sanger DNA sequencing. Another six carriers were identified amongst her relatives. Three subjects fulfilled the criteria for a definitive diagnosis of ARVC and one reached a borderline diagnosis. In conclusion, this is the first family with ARVC where a mutation in LDB3 is associated with ARVC. Next generation sequencing arises as a particular useful tool to point to new causative genes in ARVC.     

Next‐generation DNA sequencing of a Swedish malignant hyperthermia cohort

Malignant hyperthermia (MH)‐related mutations have been identified in the ryanodine receptor type 1 gene (RYR1) and in the dihydropyridine gene (CACNA1S), but about half of the patients do not have causative mutations in these genes. We wanted to study the contribution of other muscle genes to the RYR1 phenotypes. We designed a gene panel for sequence enrichment targeting 64 genes of proteins involved in the homeostasis of the striated muscle cell. Next‐generation sequencing (NGS) resulted in >50,000 sequence variants which were further analyzed by software filtering criteria to identify causative variants. In four of five patients we identified previously reported RYR1 mutations while the fifth patient did not show any candidate variant in any of the genes investigated. In two patients pathogenic variants were found in other genes known to cause a muscle disorders. All but one patient carried likely benign rare polymorphisms. The NGS technique proved convenient in identifying variants in the RYR1. However, with a clinically variable phenotype‐like MH, the pre‐selection of genes poses problems in variant interpretation.     

Biallelic HERC1 mutations in a syndromic form of overgrowth and intellectual disability

We report two Colombian siblings affected by overgrowth, intellectual disability and facial dysmorphism. Exome (via NGS) and Sanger sequencing revealed that biallelic sequence variants in a novel gene (HERC1) might be related to the disease pathogenesis. These results provide useful data for future genotype–phenotype correlations and for a molecular diagnosis of overgrowth.