Reprint of: gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.     

RECQL: a new breast cancer susceptibility gene

Identifying and characterizing novel genetic risk factors for BRCA1/2 negative breast cancers is highly relevant for early diagnosis and development of a management plan. Mutations in a number of DNA repair genes have been associated with genomic instability and development of breast and various other cancers. Whole exome sequencing efforts by 2 groups have led to the discovery in distinct populations of multiple breast cancer susceptibility mutations in RECQL, a gene that encodes a DNA helicase involved in homologous recombination repair and response to replication stress. RECQL pathogenic mutations were identified that truncated or disrupted the RECQL protein or introduced missense mutations in its helicase domain. RECQL mutations may serve as a useful biomarker for breast cancer. Targeting RECQL associated tumors with novel DNA repair inhibitors may provide a new strategy for anti-cancer therapy.     

Identification of Driving ALK Fusion Genes and Genomic Landscape of Medullary Thyroid Cancer

The genetic landscape of medullary thyroid cancer (MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14), serine/threonine kinase 11 (N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6), mutL homolog 1 (N = 4), Kiesten rat sarcoma viral oncogene homolog (N = 3) and MET proto-oncogene (N = 3). We also evaluated anaplastic lymphoma kinase (ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5’ fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK and echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target therapy.     

An abundant evolutionarily conserved CSB-PiggyBac fusion protein expressed in Cockayne syndrome

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3′ terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1–5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.     

Gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.     

MCM9 Mutations Are Associated with Ovarian Failure,Short Stature,and Chromosomal Instability

Premature ovarian failure (POF) is genetically heterogeneous and manifests as hypergonadotropic hypogonadism either as part of a syndrome or in isolation. We studied two unrelated consanguineous families with daughters exhibiting primary amenorrhea, short stature, and a 46,XX karyotype. A combination of SNP arrays, comparative genomic hybridization arrays, and whole-exome sequencing analyses identified homozygous pathogenic variants in MCM9, a gene implicated in homologous recombination and repair of double-stranded DNA breaks. In one family, the MCM9 c.1732+2T>C variant alters a splice donor site, resulting in abnormal alternative splicing and truncated forms of MCM9 that are unable to be recruited to sites of DNA damage. In the second family, MCM9 c.394C>T (p.Arg132^∗) results in a predicted loss of functional MCM9. Repair of chromosome breaks was impaired in lymphocytes from affected, but not unaffected, females in both families, consistent with MCM9 function in homologous recombination. Autosomal-recessive variants in MCM9 cause a genomic-instability syndrome associated with hypergonadotropic hypogonadism and short stature. Preferential sensitivity of germline meiosis to MCM9 functional deficiency and compromised DNA repair in the somatic component most likely account for the ovarian failure and short stature.     

PTCH1 Gene Mutations in Keratocystic Odontogenic Tumors: A Study of 43 Chinese Patients and a Systematic Review

Background

The keratocystic odontogenic tumor (KCOT) is a locally aggressive cystic jaw lesion that occurs sporadically or in association with nevoid basal cell carcinoma syndrome (NBCCS). PTCH1, the gene responsible for NBCCS, may play an important role in sporadic KCOTs. In this study, we analyzed and compared the distribution pattern of PTCH1 mutations in patients with sporadic and NBCCS-associated KCOTs.

Methods

We detected PTCH1 mutations in 14 patients with NBCCS-associated KCOTs and 29 patients with sporadic KCOTs by direct sequencing. In addition, five electronic databases were searched for studies detecting PTCH1 mutations in individuals with NBCCS-associated or sporadic KCOTs, published between January 1996 and June 2013 in English language.

Results

We identified 15 mutations in 11 cases with NBCCS-associated KCOTs and 19 mutations in 13 cases with sporadic KCOTs. In addition, a total of 204 PTCH1 mutations (187 mutations from 210 cases with NBCCS-associated and 17 mutations from 57 cases with sporadic KCOTs) were compiled from 78 published papers.

Conclusions

Our study indicates that mutations in transmembrane 2 (TM2) are closely related to the development of sporadic KCOTs. Moreover, for the early diagnosis of NBCCS, a genetic analysis of the PTCH1 gene should be included in the new diagnostic criteria.     

Targeted Exon Capture and Sequencing in Sporadic Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive degeneration of motor neurons, ultimately leading to paralysis and death. Approximately 10% of ALS cases are familial, with the remaining 90% of cases being sporadic. Genetic studies in familial cases of ALS have been extremely informative in determining the causative mutations behind ALS, especially as the same mutations identified in familial ALS can also cause sporadic disease. However, the cause of ALS in approximately 30% of familial cases and in the majority of sporadic cases remains unknown. Sporadic ALS cases represent an underutilized resource for genetic information about ALS; therefore, we undertook a targeted sequencing approach of 169 known and candidate ALS disease genes in 242 sporadic ALS cases and 129 matched controls to try to identify novel variants linked to ALS. We found a significant enrichment in novel and rare variants in cases versus controls, indicating that we are likely identifying disease associated mutations. This study highlights the utility of next generation sequencing techniques combined with functional studies and rare variant analysis tools to provide insight into the genetic etiology of a heterogeneous sporadic disease.     

MSH2 missense mutations alter cisplatin cytotoxicity and promote cisplatin-induced genome instability

Defects in the mismatch repair protein MSH2 cause tolerance to DNA damage. We report how cancer-derived and polymorphic MSH2 missense mutations affect cisplatin cytotoxicity. The chemotolerance phenotype was compared with the mutator phenotype in a yeast model system. MSH2 missense mutations display a strikingly different effect on cell death and genome instability. A mutator phenotype does not predict chemotolerance or vice versa. MSH2 mutations that were identified in tumors (Y109C) or as genetic variations (L402F) promote tolerance to cisplatin, but leave the initial mutation rate of cells unaltered. A secondary increase in the mutation rate is observed after cisplatin exposure in these strains. The mutation spectrum of cisplatin-resistant mutators identifies persistent cisplatin adduction as the cause for this acquired genome instability. Our results demonstrate that MSH2 missense mutations that were identified in tumors or as polymorphic variations can cause increased cisplatin tolerance independent of an initial mutator phenotype. Cisplatin exposure promotes drug-induced genome instability. From a mechanistical standpoint, these data demonstrate functional separation between MSH2-dependent cisplatin cytotoxicity and repair. From a clinical standpoint, these data provide valuable information on the consequences of point mutations for the success of chemotherapy and the risk for secondary carcinogenesis.     

De Novo Mutations in NALCN Cause a Syndrome Characterized by Congenital Contractures of the Limbs and Face,Hypotonia, and Developmental Delay

Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal-dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five individuals who had been putatively diagnosed with “DA2A with severe neurological abnormalities” and for whom congenital contractures of the limbs and face, hypotonia, and global developmental delay had resulted in early death in three cases; this is a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in the sodium leak channel, non-selective (NALCN) in four families affected by CLIFAHDD syndrome. We used molecular-inversion probes to screen for NALCN in a cohort of 202 distal arthrogryposis (DA)-affected individuals as well as concurrent exome sequencing of six other DA-affected individuals, thus revealing NALCN mutations in ten additional families with “atypical” forms of DA. All 14 mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that NALCN alterations nearly abolished the expression of wild-type NALCN, suggesting that alterations that cause CLIFAHDD syndrome have a dominant-negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families affected by an autosomal-recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition characterized by varied though overlapping phenotypic features, perhaps based on the type of mutation and affected protein domain(s).     

A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations

Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, “Clone-seq”, leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.     

Novel Mutations of ABCB6 Associated with Autosomal Dominant Dyschromatosis Universalis Hereditaria

Objective

Dyschromatosis universalis hereditaria (DUH) is a rare heterogeneous pigmentary genodermatosis, which was first described in 1933. The genetic cause has recently been discovered by the discovery of mutations in ABCB6. Here we investigated a Chinese family with typical features of autosomal dominant DUH and 3 unrelated patients with sporadic DUH.

Methods

Skin tissues were obtained from the proband, of this family and the 3 sporadic patients. Histopathological examination and immunohistochemical analysis of ABCB6 were performed. Peripheral blood DNA samples were obtained from 21 affected, 14 unaffected, 11 spouses in the family and the 3 sporadic patients. A genome-wide linkage scan for the family was carried out to localize the causative gene. Exome sequencing was performed from 3 affected and 1 unaffected in the family. Sanger sequencing of ABCB6 was further used to identify the causative gene for all samples obtained from available family members, the 3 sporadic patients and a panel of 455 ethnically-matched normal Chinese individuals.

Results

Histopathological analysis showed melanocytes in normal control’s skin tissue and the hyperpigmented area contained more melanized, mature melanosomes than those within the hypopigmented areas. Empty immature melanosomes were found in the hypopigmented melanocytes. Parametric multipoint linkage analysis produced a HLOD score of 4.68, with markers on chromosome 2q35-q37.2. A missense mutation (c.1663 C>A, p.Gln555Lys) in ABCB6 was identified in this family by exome and Sanger sequencing. The mutation perfectly cosegregated with the skin phenotype. An additional mutation (g.776 delC, c.459 delC) in ABCB6 was found in an unrelated sporadic patient. No mutation in ABCB6 was discovered in the other two sporadic patients. Neither of the two mutations was present in the 455 controls. Melanocytes showed positive immunoreactivity to ABCB6.

Conclusion

Our data add new variants to the repertoire of ABCB6 mutations with DUH.     

Recurrent gain-of-function USP8 mutations in Cushing's disease

Cushing''s disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing''s syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing''s disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing''s disease and provides insights into the therapeutics of this disease.     

Genetic and epigenetic alterations of the NF2 gene in sporadic vestibular schwannomas

Background

Mutations in the neurofibromatosis type 2 (NF2) tumor-suppressor gene have been identified in not only NF2-related tumors but also sporadic vestibular schwannomas (VS). This study investigated the genetic and epigenetic alterations in tumors and blood from 30 Korean patients with sporadic VS and correlated these alterations with tumor behavior.

Methodology/Principal Findings

NF2 gene mutations were detected using PCR and direct DNA sequencing and three highly polymorphic microsatellite DNA markers were used to assess the loss of heterozygosity (LOH) from chromosome 22. Aberrant hypermethylation of the CpG island of the NF2 gene was also analyzed. The tumor size, the clinical growth index, and the proliferative activity assessed using the Ki-67 labeling index were evaluated. We found 18 mutations in 16 cases of 30 schwannomas (53%). The mutations included eight frameshift mutations, seven nonsense mutations, one in-frame deletion, one splicing donor site, and one missense mutation. Nine patients (30%) showed allelic loss. No patient had aberrant hypermethylation of the NF2 gene and correlation between NF2 genetic alterations and tumor behavior was not observed in this study.

Conclusions/Significance

The molecular genetic changes in sporadic VS identified here included mutations and allelic loss, but no aberrant hypermethylation of the NF2 gene was detected. In addition, no clear genotype/phenotype correlation was identified. Therefore, it is likely that other factors contribute to tumor formation and growth.     

Genome-wide linkage in a highly consanguineous pedigree reveals two novel loci on chromosome 7 for non-syndromic familial Premature Ovarian Failure

Background

The human condition known as Premature Ovarian Failure (POF) is characterized by loss of ovarian function before the age of 40. A majority of POF cases are sporadic, but 10–15% are familial, suggesting a genetic origin of the disease. Although several causal mutations have been identified, the etiology of POF is still unknown for about 90% of the patients.

Methodology/Principal Findings

We report a genome-wide linkage and homozygosity analysis in one large consanguineous Middle-Eastern POF-affected family presenting an autosomal recessive pattern of inheritance. We identified two regions with a LOD_max of 3.26 on chromosome 7p21.1-15.3 and 7q21.3-22.2, which are supported as candidate regions by homozygosity mapping. Sequencing of the coding exons and known regulatory sequences of three candidate genes (DLX5, DLX6 and DSS1) included within the largest region did not reveal any causal mutations.

Conclusions/Significance

We detect two novel POF-associated loci on human chromosome 7, opening the way to the identification of new genes involved in the control of ovarian development and function.