Detection of DNA copy number alterations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of single nucleotide polymorphisms.

OBJECTIVES: Copy number alterations (CNAs) are frequently found in malignant tissues. Different approaches have been used for CNA detection. However, it is not easy to detect a large panel of CNA targets in heterogenous tumors. METHODS: We have developed a CNAs detection approach through quantitatively analyzed allelic imbalance by allelotyping single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the copy number changes were quantified by real-competitive PCR (rcPCR) to distinguish loss of heterozygosity (LOH) and genomic amplification. The approach was used to validate the CNA regions detected by next generation sequencing (NGS) in early-stage lung carcinoma. RESULTS: CNAs were detected in heterogeneous DNA samples where tumor DNA is present at only 10% through the SNP based allelotyping. In addition, two different types of CNAs (loss of heterozygosity and chromosome amplification) were able to be distinguished quantitatively by rcPCR. Validation on a total of 41 SNPs from the selected CNA regions showed that copy number changes did occur, and the tissues from early-stage lung carcinoma were distinguished from normal. CONCLUSIONS: CNA detection by MALDI-TOF MS can be used for validating potentially interesting genomic regions identified from next generation sequencing, and for detecting CNAs in tumor tissues consisting of a mixture of neoplastic and normal cells.

Simultaneous quantification of SMN1 and SMN2 copy numbers by MALDI-TOF mass spectrometry for spinal muscular atrophy genetic testing.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder caused by defects in the survival motor neuron 1 (SMN1) gene. Homozygous deletion of the SMN1 gene accounts for 95% of all affected SMA patients. A highly homologous gene survival motor neuron 2 (SMN2) compensates weakly with the loss of SMN1 and its copy number correlates with disease severity. METHODS: We report here the MS-CNV method combining competitive PCR and MALDI-TOF mass spectrometry for simultaneous quantification of SMN1, SMN2 and NAIP dosages. For both SMN1 and SMN2, the exon 7 and exon 8 were analyzed. MS-CNV was validated with parallel analysis by a commercial MLPA assay in two independent cohorts. RESULTS: In the first cohort of 79 blood samples containing 3 SMA patients and 5 carriers, MS-CNV results were highly concordant with MLPA analysis for the copy numbers of SMN1, SMN2 and NAIP. In the second independent and blinded cohort of 62 blood samples containing 21 SMA patients and 14 carriers, MS-CNV results were also highly concordant with MLPA. Both MS-CNV and MLPA quantified SMN1 dosages without ambiguity. CONCLUSIONS: MS-CNV can be used for carrier screening and genetic diagnosis of SMA, providing dosages information for both SMN1 and SMN2 given its accuracy and high sample processing throughput by mass spectrometric analysis.

Androgen plays an important role in regulating the synthesis of pheromone in the scent gland of muskrat.

The scent (musk) gland is an organ unique to muskrats and other scent-secreting animals, and the pheromones (musk) synthesized and secreted by the scent gland play a role in chemical communication among scent-secreting animals. The musk gland is synchronized with testicular developmental changes; however, little is known regarding androgen secretion from the testis and how this regulates pheromone synthesis and the secretion of scent. To investigate the effect of androgens on the synthesis of pheromones in the musk gland, we established a muskrat castration model by surgical removal of the testis, and analyzed the histomorphology, hormone concentration, gene expression, and changes in the chemical composition of the musk gland in castration and control groups by histomorphological analysis, Enzyme-Linked ImmunoSorbent Assay (ELISA), RNA sequencing (RNA-seq), and gas chromatography-mass spectrometry (GCMS). Histomorphological analysis results showed that after castration, muskrat gland cells underwent significant atrophy (P < 0.05). Hormone measurement results showed that there was a significant decrease in serum testosterone and muskrat musk testosterone (P < 0.05) after muskrat castration. Transcriptome sequencing results showed that 510 differentially expressed transcripts (DETs) were mainly enriched in fatty acid metabolism, terpenoid backbone biosynthesis, fatty acid degradation, PPAR signaling pathway, and fatty acid biosynthesis. GCMS results showed that macrocyclic ketones, steroids, fatty acids, alcohols, and esters in musk were significantly changed (P < 0.05). In conclusion, androgens were found to play an important function in the chemical communication exchange between muskrats through regulating pheromone synthesis in musk cells. This study provides a basis for understanding the mechanism of animal communication influenced by androgens.

Study of compositions of musks in different types secreted by forest musk deer (Moschus berezovskii).

Musk is a secretion of the forest musk deer (Moschus berezovskii). Normal musk is a brown solid secretion with a light fragrance. In this study, abnormal types of musk, namely, white and black musks, were discovered during the musk collection process. Researchers have long been concerned with the components of musk. Herein, GC-MS, headspace solid-phase microextraction (HS-SPME), and nonmetric multidimensional scaling (NMDS) were used to analyze the nonpolar organic components, volatile organic components, and sample similarities among different musks, respectively. Abundant steroid hormones and proteins were also found in the musk. The steroid hormone concentrations were detected using a radioimmunoassay (RIA). Proteins in the samples were hydrolyzed and the amino acids concentrations were detected. The steroid hormone and amino acid concentrations in white musk were significantly lower than in normal and black musks (p<0.05). The components were subjected to NMDS analysis to understand the differences in components among different types of musk, with the results suggesting that white musk was different from normal and black musks.

A Closed-Tube Nested Quantitative PCR Assay for Rapid Detection of Intron 22 Inversions in the Factor VIII Gene.

BACKGROUND: An inversion of intron 22 in the Factor VIII gene (Inv22) is the causative mutation for 45% of severe hemophilia A cases. Available methods for molecular diagnosis of Inv22 are generally tedious and not ideal for routine clinical use. METHODS: We report here a new method using a single closed-tube nested quantitative PCR (CN-qPCR) for rapid detection of Inv22. This method combines a 12-cycle long-distance PCR (LD-PCR) amplifying the int22h regions, followed by a duplex qPCR targeting two specific regions close to the int22h regions. All reagents were added to a single PCR mixture for the closed-tube assay. Sequential LD-PCR and qPCR was achieved by designing primers at substantially different melting temperatures and optimizing PCR conditions. RESULTS: Seventy-nine male hemophilia A patients of different disease severity were tested by both the CN-qPCR assay and the standard LD-PCR assay. CN-qPCR successfully made calls for all samples, whereas LD-PCR failed in eight samples. For the 71 samples where both methods made calls, the concordance was 100%. Inv22 was detected in 17 out of the 79 samples. Additionally, CN-qPCR achieved clear separation for 10 female carriers and 10 non-Inv22 females, suggesting the assay may also be useful for molecular diagnosis of female carriers. CONCLUSIONS: This new CN-qPCR method may provide a convenient and accurate F8 Inv22 test suitable for clinical use.