Formalin fixation increases deamination mutation signature but should not lead to false positive mutations in clinical practice.

Genomic analysis of cancer tissues is an essential aspect of personalized oncology treatment. Though it has been suggested that formalin fixation of patient tissues may be suboptimal for molecular studies, this tissue processing approach remains the industry standard. Therefore clinical molecular laboratories must be able to work with formalin fixed, paraffin embedded (FFPE) material. This study examines the effects of pre-analytic variables introduced by routine pathology processing on specimens used for clinical reports produced by next-generation sequencing technology. Tissue resected from three colorectal cancer patients was subjected to 2, 15, 24, and 48 hour fixation times in neutral buffered formalin. DNA was extracted from all tissues twice, once with uracil-N-glycosylase (UNG) treatment to counter deamination effects, and once without. Of note, deamination events at methylated cytosine, as found at CpG sites, remains unaffected by UNG. After extraction a two-step PCR targeted sequencing method was performed using the Illumina MiSeq and the data was analyzed via a custom-built bioinformatics pipeline, including filtration of reads with mapping quality <30. A larger baseline group of samples (n = 20) was examined to establish if there was a sample performance difference between the two DNA extraction methods, with/without UNG treatment. There was no statistical difference between sequencing performance of the two extraction methods when comparing read counts (raw, mapped, and filtered) and read quality (% mapped, % filtered). Analyzing mutation type, there was no significant difference between mutation calls until the 48 hour fixation treatment. At 48 hours there is a significant increase in C/G->T/A mutations that is not represented in DNA treated with UNG. This suggests these errors may be due to deamination events triggered by a longer fixation time. However the allelic frequency of these events remained below the limit of detection for reportable mutations in this assay (<2%). We do however recommend that suspected intratumoral heterogeneity events be verified by re-sequencing the same FFPE block.

Identification of four novel genes contributing to familial elevated plasma HDL cholesterol in humans.

While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband's family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.

Segregation of LIPG, CETP, and GALNT2 mutations in Caucasian families with extremely high HDL cholesterol.

To date, few mutations are described to underlie highly-elevated HDLc levels in families. Here we sequenced the coding regions and adjacent sequence of the LIPG, CETP, and GALNT2 genes in 171 unrelated Dutch Caucasian probands with HDLc≥90th percentile and analyzed segregation of mutations with lipid phenotypes in family members. In these probands, mutations were most frequent in LIPG (12.9%) followed by GALNT2 (2.3%) and CETP (0.6%). A total of 6 of 10 mutations in these three genes were novel (60.0%), and mutations segregated with elevated HDLc in families. Interestingly, the LIPG mutations N396S and R476W, which usually result in elevated HDLc, were unexpectedly found in 6 probands with low HDLc (i.e., ≤10th percentile). However, 5 of these probands also carried mutations in ABCA1, LCAT, or LPL. Finally, no CETP and GALNT2 mutations were found in 136 unrelated probands with low HDLc. Taken together, we show that rare coding and splicing mutations in LIPG, CETP, and GALNT2 are enriched in persons with hyperalphalipoproteinemia and segregate with elevated HDLc in families. Moreover, LIPG mutations do not overcome low HDLc in individuals with ABCA1 and possibly LCAT and LPL mutations, indicating that LIPG affects HDLc levels downstream of these proteins.

Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.

Juvenile hemochromatosis is an early-onset autosomal recessive disorder of iron overload resulting in cardiomyopathy, diabetes and hypogonadism that presents in the teens and early 20s (refs. 1,2). Juvenile hemochromatosis has previously been linked to the centromeric region of chromosome 1q (refs. 3-6), a region that is incomplete in the human genome assembly. Here we report the positional cloning of the locus associated with juvenile hemochromatosis and the identification of a new gene crucial to iron metabolism. We finely mapped the recombinant interval in families of Greek descent and identified multiple deleterious mutations in a transcription unit of previously unknown function (LOC148738), now called HFE2, whose protein product we call hemojuvelin. Analysis of Greek, Canadian and French families indicated that one mutation, the amino acid substitution G320V, was observed in all three populations and accounted for two-thirds of the mutations found. HFE2 transcript expression was restricted to liver, heart and skeletal muscle, similar to that of hepcidin, a key protein implicated in iron metabolism. Urinary hepcidin levels were depressed in individuals with juvenile hemochromatosis, suggesting that hemojuvelin is probably not the hepcidin receptor. Rather, HFE2 seems to modulate hepcidin expression.