Meta-analysis of gross insertions causing human genetic disease: novel mutational mechanisms and the role of replication slippage

Although gross insertions (>20 bp) comprise <1% of disease-causing mutations, they nevertheless represent an important category of pathological lesion. In an attempt to study these insertions in a systematic way, 158 gross insertions ranging in size between 21 bp and approximately 10 kb were identified using the Human Gene Mutation Database (www.hgmd.org). A careful meta-analytical study revealed extensive diversity in terms of the nature of the inserted DNA sequence and has provided new insights into the underlying mutational mechanisms. Some 70% of gross insertions were found to represent sequence duplications of different types (tandem, partial tandem, or complex). Although most of the tandem duplications were explicable by simple replication slippage, the three complex duplications appear to result from multiple slippage events. Some 11% of gross insertions were attributable to nonpolyglutamine repeat expansions (including octapeptide repeat expansions in the prion protein gene [PRNP] and polyalanine tract expansions) and evidence is presented to support the contention that these mutations are also caused by replication slippage rather than by unequal crossing over. Some 17% of gross insertions, all >or=276 bp in length, were found to be due to LINE-1 (L1) retrotransposition involving different types of element (L1 trans-driven Alu, L1 direct, and L1 trans-driven SVA). A second example of pathological mitochondrial-nuclear sequence transfer was identified in the USH1C gene but appears to arise via a novel mechanism, trans-replication slippage. Finally, evidence for another novel mechanism of human genetic disease, involving the possible capture of DNA oligonucleotides, is presented in the context of a 26-bp insertion into the ERCC6 gene.     

Genomic characterization of KIR2DL4 in families and unrelated individuals reveals extensive diversity in exon and intron sequences including a common frameshift variation occurring in several alleles

The KIR2DL4 gene including a portion of exon 1 through exon 9 was sequenced from two families and eight cell lines from the International Histocompatibility Workshop (IHWS). Two known alleles and eight variants were detected. Overall, there were five synonymous and three non-synonymous changes when the variants were compared to the coding sequences of the most closely related known alleles plus a common frameshift change in five of the variant alleles. Alignment of the new variants with all known alleles showed that the regions encoding the extracellular region and the cytoplasmic tail were the most polymorphic. Two non-synonymous changes, P146H and L161V, occurred in an extracellular immunoglobulin-like domain. Five of the eight variants had a single adenine deletion in the exon encoding the transmembrane region, potentially resulting in a truncated protein lacking the cytoplasmic tail. The distribution of the deletion variant among many KIR2DL4 alleles may explain the high frequency of this variation in the population. Four of the eight consanguineous IHWS cell lines were found to be heterozygous for KIR2DL4 carrying two alleles that differed from one another by a few nucleotide substitutions. Analysis of intron sequences in the families revealed the nature and distribution of interspersed repeat elements which comprise 46% of the KIR2DL4 nucleotide sequence and consist of 12 elements including six SINEs (13.73% of the total length), one LINE (12.41%), and five LTR elements (19.51%). The results revealed the presence of extensive diversity in the KIR2DL4 gene. This is the first extensive report providing both exon and intron data in related individuals.     

Response of Human Insulinoma Cells to Extracellular Calcium Is Different from Normal B Cells

The preoperative determination of thelocalization of a small insulinoma is sometimesdifficult using routine imaging techniques. We have usedthe selective arterial calcium injection (SACI) test todetermine the location of the tumor preoperatively. Thepathophysiologic basis of the SACI test is based on theresponsiveness of insulinomas to calcium injected intothe feeding artery. In this study, we demonstrated the in vitro response of the insulinoma cellsto the extracellular calcium challenge by usingprimary-cultured insulinoma cells. Human insulinomacells were obtained from three patients. MIN6 cells(normal pancreatic B cells) were used as a control;their insulin response to various stimuli resembles thatof normal B cells. The insulin secretory dynamics inresponse to extracellular calcium were observed using a perfusion system. Second, the change ofthe concentration of cytosolic free calcium([Ca²⁺]_i) was monitored byfluorometry using fura-2/AM. When the concentration ofextracellular calcium ([Ca²⁺]_o) was changed from 2.54 mM to 10 mM, insulinsecretion from the insulinoma cells was markedlyincreased within 6 min (10- to 18-fold at maximum), andrapidly returned to the basal level; at the same time, [Ca²⁺]_i was immediatelyelevated and reached a peak within 1 min. In contrast,in the MIN6 cells, the insulin secretion and [Ca2+]iwere not significantly changed when[Ca²⁺]_o was switched to 10 mM. The results of these in vitro experiments agreedwith the clinical results of the SACI test. The positiveresponse of the insulinoma to the SACI test is probablydue to the different response of insulinoma cells to the extracellular calcium challengecompared with normal B cells. The role of[Ca²⁺]_i may be important in themechanism underlying the SACI test.     

Revealing the human mutome

Chen JM, Férec C, Cooper DN. Revealing the human mutome. The number of known mutations in human nuclear genes, underlying or associated with human inherited disease, has now exceeded 100,000 in more than 3700 different genes (Human Gene Mutation Database). However, for a variety of reasons, this figure is likely to represent only a small proportion of the clinically relevant genetic variants that remain to be identified in the human genome (the ‘mutome’). With the advent of next‐generation sequencing, we are currently witnessing a revolution in medical genetics. In particular, whole‐genome sequencing (WGS) has the potential to identify all disease‐causing or disease‐associated DNA variants in a given individual. Here, we use examples of recent advances in our understanding of mutational/pathogenic mechanisms to guide our thinking about possible locations outwith gene‐coding sequences for those disease‐causing or disease‐associated variants that are likely so often to have been overlooked because of the inadequacy of current mutation screening protocols. Such considerations are important not only for improving mutation‐screening strategies but also for enhancing the interpretation of findings derived from genome‐wide association studies, whole‐exome sequencing and WGS. An improved understanding of the human mutome will not only lead to the development of improved diagnostic testing procedures but should also improve our understanding of human genome biology.