DNA damage response gene mutations and inherited susceptibility to breast cancer

Abstract Breast cancer is the most common malignancy in women and it is strongly influenced by hereditary risk factors. So far, most of the breast cancer-associated genes, including BRCA1/2, have been identified among those that encode proteins involved in DNA damage response (DDR) pathways. However, known genetic risk factors explain less than half of the familial risk of breast cancer. Identification of novel genes and mutations that predispose to breast cancer is important for the understanding of the mechanisms that contribute to the disease development and also for the identification of those individuals who are at high risk. The first aim of this study was to resolve the complementation groups of Finnish patients with Fanconi anemia (FA), which is a rare genetic disease caused by defects in a specific DDR pathway, and to study the role of the causative gene mutations in breast cancer predisposition. The second aim of this study was to identify novel breast cancer susceptibility genes and alleles by targeted next-generation sequencing (NGS) of multiple (~800) DDR related genes. In both approaches, the identified gene mutations were subjected to case-control association analysis utilizing DNA samples of over 1,000 breast cancer cases and 1,000 healthy controls. Investigation of the Finnish FA patients revealed six different disease-causing mutations in three different genes (FANCA, FANCG and FANCI). All of the studied mutations were recurrent in the Finnish population but did not associate with breast cancer. Targeted NGS identified three novel potential breast cancer susceptibility genes. A significant enrichment of TEX15 c.7253dupT and FANCD2 c.2715+1G>A mutations was observed among the hereditary breast cancer cases (P = 0.018 and P = 0.036, respectively). The strongest evidence was found for a Finnish founder mutation in MCPH1 (c.904_916del), which associated with breast cancer susceptibility both in familial (P = 0.003, OR 8.3) and unselected (P = 0.016, OR 3.3) patient cohorts. The tumor suppressive function of MCPH1 was indicated by the loss of the wild-type allele of MCPH1 in 40% of the studied carrier tumors. Furthermore, carriers exhibited a significant increase in genomic instability measured by spontaneous chromosomal rearrangements in peripheral blood lymphocytes.Tiivistelmä Rintasyöpä on naisten yleisin syöpä. Sairastumisriskiin vaikuttavat voimakkaasti perinnölliset alttiustekijät, ja suurin osa tähän asti tunnistetuista rintasyöpäalttiusgeeneistä, kuten BRCA1/2, koodaa DNA-vauriovasteessa (DDR) toimivia proteiineja. Tunnistetut tekijät selittävät yhä kuitenkin vain alle puolet rintasyövän perinnöllisestä alttiudesta. Uusien alttiusgeenien tunnistaminen on tärkeää rintasyövän patomekanismien ymmärtämiseksi sekä korkean rintasyöpäriskin omaavien henkilöiden tunnistamiseksi. Tämän tutkimuksen tarkoituksena oli määrittää viallisesta DDR-signaalinsiirtoreitistä johtuvan Fanconin anemian (FA) komplementaatioryhmät suomalaisilta FA-potilailta sekä tutkia sairauden taustalla olevien geenimutaatioden yhteyttä rintasyöpäriskiin. Uusia alttiusgeenejä etsittiin myös kohdennetulla uuden sukupolven sekvensointimenetelmällä, jonka avulla tutkittiin yhtäaikaisesti n. 800 DDR-geeniä. Molemmilla lähestymistavoilla tunnistettujen geenimuutosten yhteyttä rintasyöpään selvitettiin tapaus-verrokkitutkimuksen avulla, jossa tutkittiin DNA-näytteitä yli tuhannelta rintasyöpäpotilaalta sekä yli tuhannelta terveeltä henkilöltä. Suomalaisten FA-potilaiden geenimuutoksia selvittävässä tutkimuksessa tunnistettiin yhteensä kuusi mutaatiota kolmessa eri geenissä (FANCA, FANCG ja FANCI). Kaikki tutkimuksessa tunnistetut mutaatiot olivat toistuvia suomalaisessa väestössä, mutta merkitsevää assosiaatiota näiden mutaatioiden ja rintasyöpäalttiuden välillä ei havaittu. DDR-geenien sekvensoinnin avulla tunnistettiin kolme uutta mahdollista rintasyöpäalttiusgeeniä. Tutkimuksessa havaittiin TEX15 c.7253dupT ja FANCD2 c.2715+1G>A mutaatioiden rikastuminen perinnöllisessä rintasyöpäaineistossa (P = 0.018 ja P = 0.036). Merkittävin yhteys rintasyöpäalttiuden kanssa todettiin MCPH1-geenin perustajamutaatiolle (c.904_916del). Tämä mutaatio assosioitui rintasyöpäalttiuden kanssa sekä perinnöllisessä (P = 0.003, OR 8.3) että valikoimattomassa potilasaineistossa (P = 0.016, OR 3.3). Useissa mutaatiokantajien tuumoreissa (40 %) normaali MCPH1 vastinalleeli oli hävinnyt, mikä viittaisi siihen, että MCPH1 toimii tuumorisuppressorina. Mutaatiokantajilla todettiin myös kohonnut määrä kromosomaalisia muutoksia veren periferaalisissa lymfosyyteissä, mahdollisesti kohonneeseen genomiseen epävakauteen liittyen

Uudet long-read-teknologiat:kohti tarkennettua genomitietoa perinnöllisistä sairauksista ja syövistä

Tiivistelmä Uusien molekyylibiologisten menetelmien kehityksellä ja laajamittaisella hyödyntämisellä on avainasema eri sairauksien taustalla vaikuttavan geneettisen vaihtelun tutkimisessa. Uuden sukupolven sekvensointimenetelmät ovat mahdollistaneet lukuisien tautigeenien tunnistamisen, ja kustannustehokkuutensa ansioista ne ovat tulleet tärkeäksi osaksi myös kliinistä rutiinidiagnostiikkaa. On silti tärkeää tiedostaa näiden menetelmien puutteet erityisesti genomin rakenteellisten muutosten havaitsemisessa, jossa hyödynnetään edelleen pääasiassa tavanomaisia sytogeneettisiä menetelmiä. Viimeaikaisen teknologisen kehityksen myötä uudet long-read-menetelmät voivat mahdollistaa entistä tarkemman ja kattavamman genomitiedon muodostamisen sekä korvata nykyisiä tutkimusmenetelmiä tulevaisuudessa

Long-read sequencing emerging in medical genetics

Abstract The wide implementation of next-generation sequencing (NGS) technologies has revolutionized the field of medical genetics. However, the short read lengths of currently used sequencing approaches pose a limitation for the identification of structural variants, sequencing repetitive regions, phasing of alleles and distinguishing highly homologous genomic regions. These limitations may significantly contribute to the diagnostic gap in patients with genetic disorders who have undergone standard NGS, like whole exome or even genome sequencing. Now, the emerging long-read sequencing (LRS) technologies may offer improvements in the characterization of genetic variation and regions that are difficult to assess with the prevailing NGS approaches. LRS has so far mainly been used to investigate genetic disorders with previously known or strongly suspected disease loci. While these targeted approaches already show the potential of LRS, it remains to be seen whether LRS technologies can soon enable true whole genome sequencing routinely. Ultimately, this could allow the de novo assembly of individual whole genomes used as a generic test for genetic disorders. In this article, we summarize the current LRS-based research on human genetic disorders and discuss the potential of these technologies to facilitate the next major advancements in medical genetics

Optical genome mapping as an alternative to FISH-based cytogenetic assessment in chronic lymphocytic leukemia

Abstract The fluorescence in situ hybridization (FISH) technique plays an important role in the risk stratification and clinical management of patients with chronic lymphocytic leukemia (CLL). For genome-wide analysis, FISH needs to be complemented with other cytogenetic methods, including karyotyping and/or chromosomal microarrays. However, this is often not feasible in a diagnostic setup. Optical genome mapping (OGM) is a novel technique for high-resolution genome-wide detection of structural variants (SVs), and previous studies have indicated that OGM could serve as a generic cytogenetic tool for hematological malignancies. Herein, we report the results from our study evaluating the concordance of OGM and standard-of-care FISH in 18 CLL samples. The results were fully concordant between these two techniques in the blinded comparison. Using in silico dilution series, the lowest limit of detection with OGM was determined to range between 3 and 9% variant allele fractions. Genome-wide analysis by OGM revealed additional (>1 Mb) aberrations in 78% of the samples, including both unbalanced and balanced SVs. Importantly, OGM also enabled the detection of clinically relevant complex karyotypes, undetectable by FISH, in three samples. Overall, this study demonstrates the potential of OGM as a first-tier cytogenetic test for CLL and as a powerful tool for genome-wide SV analysis

Evaluating the role of CHEK2 p.(Asp438Tyr) allele in inherited breast cancer predisposition

Abstract CHEK2 is a well-established breast cancer susceptibility gene. The most frequent pathogenic CHEK2 variant is 1100delC, a loss-of-function mutation conferring 2-fold risk for breast cancer. This gene also harbors other rare variants encountered in the clinical gene panels for hereditary cancer. One of these is CHEK2 c.1312 G > T, p.(Asp438Tyr) in the kinase domain of the protein, but due to its rarity its clinical significance for breast cancer predisposition has remained unclear. Here, we tested the prevalence of CHEK2 p.(Asp438Tyr) allele showing enrichment in the Northern Finnish population, in a total of 2284 breast cancer patients from this geographical region. Genotyping was performed for DNA samples extracted from peripheral blood using high-resolution melt analysis. Fourteen CHEK2 p.(Asp438Tyr) carriers were identified (14/2284, 0.6%, P = 0.67): two in the cohort of breast cancer cases with the indication of inherited disease susceptibility (2/281, 0.7%, P = 1.00) and twelve in the breast cancer cohort unselected for the family history of disease and age at disease onset (12/2003, 0.6%, P = 0.66). This frequency did not differ from the frequency in the general population (10/1299, 0.8%). No CHEK2 p.(Asp438Tyr) homozygotes were identified. Our results indicate that CHEK2 p.(Asp438Tyr) carriers do not have an increased risk for breast cancer and the classification of the CHEK2 p.(Asp438Tyr) variant can be changed from the variant of uncertain significance (VUS) to likely benign for breast cancer

Truncating TINF2 p.Tyr312Ter variant and inherited breast cancer susceptibility

Abstract TINF2 is a critical subunit of the shelterin complex, which protects and maintains the length of telomeres. Pathogenic missense and truncating TINF2 mutations are causative for dyskeratosis congenita (DC), a rare, dominantly inherited bone marrow failure syndrome characterized by mucocutaneous abnormalities and cancer predisposition. Recent reports indicate that specific TINF2 truncating mutations act as high penetrance cancer predisposition alleles outside DC context, including breast cancer in their tumor spectrum. Here, we have evaluated the role of germline mutations in TINF2 and other shelterin genes in inherited breast cancer susceptibility using exome sequencing data from 98 Northern Finnish breast cancer cases with indication of inherited disease predisposition as a discovery cohort. A single protein truncating variant, TINF2 p.Tyr312Ter, was identified in one of the cases (1/98), and four more carriers were observed in the subsequently genotyped unselected breast cancer cohort (4/1904). None of the carriers were reported to have DC. TINF2 p.Tyr312Ter resulted in stable short form of mRNA transcript, and normal telomere length has been indicated by a recent report. Although recurrent in cases (total of 5/2095), TINF2 p.Tyr312Ter is also present in Finnish population controls (8/12,517), and the observed 4-fold higher frequency in cases falls at most into the range of moderate breast cancer risk alleles (OR 3.74, 95% CI 1.22–11.45, p = 0.029). Current results indicate that not all TINF2 truncating variants are high cancer risk alleles and add further evidence that different TINF2 mutations can have very diverse effects on the disease phenotype

Evaluating the role of NTHL1 p.Q90* allele in inherited breast cancer predisposition

Abstract Background: Rare protein truncating variants of NTHL1 gene are causative for the recently described, recessively inherited NTHL1 tumor syndrome that is characterized by an increased lifetime risk for colorectal cancer, colorectal polyposis, and breast cancer. Although there is strong evidence for breast cancer being a part of the cancer spectrum in these families, the role of pathogenic NTHL1 variants in breast cancer susceptibility in general population remains unclear. Methods: We tested the prevalence of NTHL1 nonsense variant c.268C>T, p.Q90*, which is the major allele in NTHL1 families and also shows enrichment in the Finnish population, in a total of 1333 breast cancer patients. Genotyping was performed for DNA samples extracted from peripheral blood by using high‐resolution melt analysis. Results: Sixteen NTHL1 p.Q90* heterozygous carriers were identified (1.2%, p = 0.61): 5 in hereditary cohort (n = 234, 2.1%, p = 0.39) and 11 in unselected cohort (n = 1099, 1.0%, p = 0.36). This frequency is equal to that in the general population (19/1324, 1.4%). No NTHL1 p.Q90* homozygotes were identified. Conclusion: Our results indicate that NTHL1 p.Q90* heterozygous carriers do not have an increased risk for breast cancer and that the variant is unlikely to be a significant contributor to breast cancer risk at the population level

Tumor suppressor MCPH1 regulates gene expression profiles related to malignant conversion and chromosomal assembly

Abstract Strong inherited predisposition to breast cancer is estimated to cause about 5–10% of all breast cancer cases. As the known susceptibility genes, such as BRCA1 and BRCA2, explain only a fraction of this, additional predisposing genes and related biological mechanisms are actively being searched for. We have recently identified a recurrent MCPH1 germline mutation, p.Arg304ValfsTer3, as a breast cancer susceptibility allele. MCPH1 encodes a multifunctional protein involved in maintenance of genomic integrity and it is also somatically altered in various cancer types, including breast cancer. Additionally, biallelic MCPH1 mutations are causative for microcephaly and at cellular level premature chromosome condensation. To study the molecular mechanisms leading to cancer predisposition and malignant conversion, here we have modeled the effect of MCPH1 p.Arg304ValfsTer3 mutation using gene‐edited MCF10A breast epithelial cells. As a complementary approach, we also sought for additional potential cancer driver mutations in MCPH1 p.Arg304ValfsTer3 carrier breast tumors. We show that mutated MCPH1 de‐regulates transcriptional programs related to invasion and metastasis and leads to downregulation of histone genes. These global transcriptional changes are mirrored by significantly increased migration and invasion potential of the cells as well as abnormal chromosomal condensation both before and after mitosis. These findings provide novel molecular insights to MCPH1 tumor suppressor functions and establish a role in regulation of transcriptional programs related to malignant conversion and chromosomal assembly. The MCPH1 p.Arg304ValfsTer3 carrier breast tumors showed recurrent tumor suppressor gene TP53 mutations, which were also significantly over‐represented in breast tumors with somatically inactivated MCPH1

Exome sequencing identified rare recurrent copy number variants and hereditary breast cancer susceptibility.

Copy number variants (CNVs) are a major source of genetic variation and can disrupt genes or affect gene dosage. They are known to be causal or underlie predisposition to various diseases. However, the role of CNVs in inherited breast cancer susceptibility has not been thoroughly investigated. To address this, we performed whole-exome sequencing based analysis of rare CNVs in 98 high-risk Northern Finnish breast cancer cases. After filtering, selected candidate alleles were validated and characterized with a combination of orthogonal methods, including PCR-based approaches, optical genome mapping and long-read sequencing. This revealed three recurrent alterations: a 31 kb deletion co-occurring with a retrotransposon insertion (delins) in RAD52, a 13.4 kb deletion in HSD17B14 and a 64 kb partial duplication of RAD51C. Notably, all these genes encode proteins involved in pathways previously identified as essential for breast cancer development. Variants were genotyped in geographically matched cases and controls (altogether 278 hereditary and 1983 unselected breast cancer cases, and 1229 controls). The RAD52 delins and HSD17B14 deletion both showed significant enrichment among cases with indications of hereditary disease susceptibility. RAD52 delins was identified in 7/278 cases (2.5%, P = 0.034, OR = 2.86, 95% CI = 1.10-7.45) and HSD17B14 deletion in 8/278 cases (2.9%, P = 0.014, OR = 3.28, 95% CI = 1.31-8.23), the frequency of both variants in the controls being 11/1229 (0.9%). This suggests a role for RAD52 and HSD17B14 in hereditary breast cancer susceptibility. The RAD51C duplication was very rare, identified only in 2/278 of hereditary cases and 2/1229 controls (P = 0.157, OR = 4.45, 95% CI = 0.62-31.70). The identification of recurrent CNVs in these genes, and especially the relatively high frequency of RAD52 and HSD17B14 alterations in the Finnish population, highlights the importance of studying CNVs alongside single nucleotide variants when searching for genetic factors underlying hereditary disease predisposition