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

TiivistelmäUusien 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.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

ATM c.7570G>C is a high-risk allele for breast cancer

AbstractATM is generally described as a moderate-risk breast cancer susceptibility gene. However, some of ATM variants might encounter higher risk. ATM c.7570G>C, p.Ala2524Pro, (rs769142993) is a pathogenic Finnish founder variant causative for recessively inherited ataxia-telangiectasia. At cellular level, it has been reported to have a dominant-negative effect. ATM c.7570G>C has recurrently been described in Finnish breast cancer families and unselected case cohorts collected from different parts of the country, but the rarity of the allele (MAF 0.0002772 in Finns) and lack of confirming segregation analyses have prevented any conclusive risk estimates. Here, we describe seven families from genetic counseling units with ATM c.7570G>C variant showing co-segregation with breast cancer. Further analysis of the unselected breast cancer cohort from Northern Finland (n = 1822), a geographical region previously indicated to have enrichment of the variant, demonstrated that c.7570G>C significantly associates with breast cancer, and the risk is estimated as high (odds ratio [OR] = 8.5, 95% confidence interval [CI] = 1.04-62.46, P = .018). Altogether, these results place ATM c.7570G>C variant among the high-risk alleles for breast cancer, which should be taken into consideration in genetic counseling.Abstract ATM is generally described as a moderate-risk breast cancer susceptibility gene. However, some of ATM variants might encounter higher risk. ATM c.7570G>C, p.Ala2524Pro, (rs769142993) is a pathogenic Finnish founder variant causative for recessively inherited ataxia-telangiectasia. At cellular level, it has been reported to have a dominant-negative effect. ATM c.7570G>C has recurrently been described in Finnish breast cancer families and unselected case cohorts collected from different parts of the country, but the rarity of the allele (MAF 0.0002772 in Finns) and lack of confirming segregation analyses have prevented any conclusive risk estimates. Here, we describe seven families from genetic counseling units with ATM c.7570G>C variant showing co-segregation with breast cancer. Further analysis of the unselected breast cancer cohort from Northern Finland (n = 1822), a geographical region previously indicated to have enrichment of the variant, demonstrated that c.7570G>C significantly associates with breast cancer, and the risk is estimated as high (odds ratio [OR] = 8.5, 95% confidence interval [CI] = 1.04-62.46, P = .018). Altogether, these results place ATM c.7570G>C variant among the high-risk alleles for breast cancer, which should be taken into consideration in genetic counseling

Recurrent CYP2C19 deletion allele is associated with triple-negative breast cancer

Background: Using a genome-wide approach, we have previously observed an increase in the frequency of rare copy number variants (CNVs) in familial and early-onset breast cancer cases when compared to controls. Moreover, the biological networks of the CNV disrupted genes differed between the two groups. Here, six of the previously observed CNVs were selected for further investigation. Four of these were singletons and disturbed the following genes: DCLRE1C, CASP3, DAB2IP and ITGA9, encoding proteins that are part of the TP53 and β-estradiol centered network. The two others were recurrent alleles and disrupted CDH19 and CYP2C19 genes. Of these, CDH19 encodes a cadherin functioning as a cell-cell adhesion receptor and CYP2C19 a CYP450 enzyme with a major function in estrogen catabolism. Methods: The exact breakpoints of the six previously observed CNV deletion alleles were defined by using qPCR, nested PCR and sequencing. The prevalence of these CNVs was investigated in 842 Northern Finnish breast cancer cases, unselected for family history of cancer and age at disease onset, as well as in 497 healthy female controls by using multiplex PCR. Also the association of the relatively common CDH19 and CYP2C19 deletion alleles with different clinical parameters was studied. Results: No significant differences in the carrier frequencies between cases and controls were found for any of the studied CNVs. However, the deletion in CYP2C19 showed a significant association with triple-negative breast cancer (p = 0.021). Conclusion: Our results indicate that inherited changes in CYP2C19 gene participating in estrogen catabolism have an influence on the molecular subtype of breast cancer.publishedVersionPeer reviewe

Exome sequencing identifies a recurrent variant in <em>SERPINA3</em> associating with hereditary susceptibility to breast cancer

AbstractBackground: Breast cancer is strongly influenced by hereditary risk factors. Yet, the known susceptibility genes and genomic loci explain only about half of the familial component of the disease. To identify novel breast cancer predisposing gene defects, here we have performed massive parallel sequencing for Northern Finnish breast cancer cases.Methods: Ninety-eight breast cancer cases with indication of hereditary disease susceptibility were exome sequenced. Data filtering strategy focused on predictably deleterious rare variants that were still enriched in the sequenced cohort. Findings were confirmed with additional, geographically matched breast cancer cohorts.Results: A recurrent heterozygous splice acceptor variant, c.918-1G>C, in SERPINA3, was identified, and it was significantly enriched both in the hereditary (6/201, 3.0%, p = 0.006, OR 5.1, 95% CI 1.7–14.8) and unselected breast cancer cohort (26/1569, 1.7%, p = 0.009, OR 2.8, 95% CI 1.3–6.2). SERPINA3 c.918-1G>C carriers were also significantly more likely to have a rare tumor subtype, medullary breast cancer, than the non-carriers (4/26, 15.4%, p = 0.000014, OR 42.9, 95% CI 11.7–157.1).Conclusion: These findings demonstrate that c.918-1G>C germline variant in SERPINA3 gene, encoding a member of the serine protease inhibitor class, is a novel breast cancer predisposing allele.Abstract Background: Breast cancer is strongly influenced by hereditary risk factors. Yet, the known susceptibility genes and genomic loci explain only about half of the familial component of the disease. To identify novel breast cancer predisposing gene defects, here we have performed massive parallel sequencing for Northern Finnish breast cancer cases. Methods: Ninety-eight breast cancer cases with indication of hereditary disease susceptibility were exome sequenced. Data filtering strategy focused on predictably deleterious rare variants that were still enriched in the sequenced cohort. Findings were confirmed with additional, geographically matched breast cancer cohorts. Results: A recurrent heterozygous splice acceptor variant, c.918-1G>C, in SERPINA3, was identified, and it was significantly enriched both in the hereditary (6/201, 3.0%, p = 0.006, OR 5.1, 95% CI 1.7–14.8) and unselected breast cancer cohort (26/1569, 1.7%, p = 0.009, OR 2.8, 95% CI 1.3–6.2). SERPINA3 c.918-1G>C carriers were also significantly more likely to have a rare tumor subtype, medullary breast cancer, than the non-carriers (4/26, 15.4%, p = 0.000014, OR 42.9, 95% CI 11.7–157.1). Conclusion: These findings demonstrate that c.918-1G>C germline variant in SERPINA3 gene, encoding a member of the serine protease inhibitor class, is a novel breast cancer predisposing allele

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

AbstractCHEK2 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.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

Recurrent ocular toxoplasmosis is associated with interferon-gamma deficiency possibly due to genetic origin

Abstract Objective: Ocular toxoplasmosis (OT) can cause posterior uveitis; causes of recurrent OT are not well understood. We explored clinical, immunological and genetic properties associated with recurrent OT. Methods and analysis: A recurrent OT patient population (n=9) was identified. Clinical history, ophthalmological findings and immunological properties were assessed. B and T cell immunophenotyping including interferon-gamma (IFN-γ) responses were analysed. An analysis of 592 immunodeficiency genes was performed. Results: The patients experienced 2–7 OT episodes (average 3.7). The first episode occurred at an average of 23.8 (SD 10.1) years of age. All patients had anterior uveitis, vitritis and various fundus lesions of OT. The patients had lymphocyte maturation abnormalities; the proportion of naive CD4+CD45RA+CCR7+ T cells was high in 5/9 cases, and the percentage of CD4+CD45RA−CCR7− T effector memory cells was reduced in 7/9 cases. An increased percentage of CD19+CD38lowCD21low activated B cells was observed in 5/9 cases. IFN-γ response was reduced in CD4+ (8.45±4.17 vs 21.27±11.0, p=0.025) and CD8+ (39.0±9.9 vs 18.1±18.1, p=0.017) T cells. Genetic analysis revealed several potentially harmful variants in immunologically active ERCC3, MANBA, IRF4, HAVCR2, CARMIL2, CD247, MPO, C2 and CD40 genes. Conclusion: Our recurrent OT cases had deviations in lymphocyte maturation and IFN-γ responses possibly caused by genetic reasons. However, limitations of our study include failure to identify uniform genetic mechanisms. In addition, we cannot rule out the possibility that the immunological abnormalities can be triggered by chronic toxoplasmosis. Despite the limitations, our findings contribute to the understanding of ocular immunity and development of recurrent OT.Abstract Objective: Ocular toxoplasmosis (OT) can cause posterior uveitis; causes of recurrent OT are not well understood. We explored clinical, immunological and genetic properties associated with recurrent OT. Methods and analysis: A recurrent OT patient population (n=9) was identified. Clinical history, ophthalmological findings and immunological properties were assessed. B and T cell immunophenotyping including interferon-gamma (IFN-γ) responses were analysed. An analysis of 592 immunodeficiency genes was performed. Results: The patients experienced 2–7 OT episodes (average 3.7). The first episode occurred at an average of 23.8 (SD 10.1) years of age. All patients had anterior uveitis, vitritis and various fundus lesions of OT. The patients had lymphocyte maturation abnormalities; the proportion of naive CD4+CD45RA+CCR7+ T cells was high in 5/9 cases, and the percentage of CD4+CD45RA−CCR7− T effector memory cells was reduced in 7/9 cases. An increased percentage of CD19+CD38lowCD21low activated B cells was observed in 5/9 cases. IFN-γ response was reduced in CD4+ (8.45±4.17 vs 21.27±11.0, p=0.025) and CD8+ (39.0±9.9 vs 18.1±18.1, p=0.017) T cells. Genetic analysis revealed several potentially harmful variants in immunologically active ERCC3, MANBA, IRF4, HAVCR2, CARMIL2, CD247, MPO, C2 and CD40 genes. Conclusion: Our recurrent OT cases had deviations in lymphocyte maturation and IFN-γ responses possibly caused by genetic reasons. However, limitations of our study include failure to identify uniform genetic mechanisms. In addition, we cannot rule out the possibility that the immunological abnormalities can be triggered by chronic toxoplasmosis. Despite the limitations, our findings contribute to the understanding of ocular immunity and development of recurrent OT

PALB2-mutated human mammary cells display a broad spectrum of morphological and functional abnormalities induced by increased TGFβ signaling

Abstract Heterozygous mutations in any of three major genes, BRCA1, BRCA2 and PALB2, are associated with high-risk hereditary breast cancer susceptibility frequently seen as familial disease clustering. PALB2 is a key interaction partner and regulator of several vital cellular activities of BRCA1 and BRCA2, and is thus required for DNA damage repair and alleviation of replicative and oxidative stress. Little is however known about how PALB2-deficiency affects cell function beyond that, especially in the three-dimensional setting, and also about its role during early steps of malignancy development. To answer these questions, we have generated biologically relevant MCF10A mammary epithelial cell lines with mutations that are comparable to certain clinically important PALB2 defects. We show in a non-cancerous background how both mono- and biallelically PALB2-mutated cells exhibit gross spontaneous DNA damage and mitotic aberrations. Furthermore, PALB2-deficiency disturbs three-dimensional spheroid morphology, increases the migrational capacity and invasiveness of the cells, and broadly alters their transcriptome profiles. TGFβ signaling and KRT14 expression are enhanced in PALB2-mutated cells and their inhibition and knock down, respectively, lead to partial restoration of cell functions. KRT14-positive cells are also more abundant with DNA damage than KRT14-negative cells. The obtained results indicate comprehensive cellular changes upon PALB2 mutations, even in the presence of half dosage of wild type PALB2 and demonstrate how PALB2 mutations may predispose their carriers to malignancy.Abstract Heterozygous mutations in any of three major genes, BRCA1, BRCA2 and PALB2, are associated with high-risk hereditary breast cancer susceptibility frequently seen as familial disease clustering. PALB2 is a key interaction partner and regulator of several vital cellular activities of BRCA1 and BRCA2, and is thus required for DNA damage repair and alleviation of replicative and oxidative stress. Little is however known about how PALB2-deficiency affects cell function beyond that, especially in the three-dimensional setting, and also about its role during early steps of malignancy development. To answer these questions, we have generated biologically relevant MCF10A mammary epithelial cell lines with mutations that are comparable to certain clinically important PALB2 defects. We show in a non-cancerous background how both mono- and biallelically PALB2-mutated cells exhibit gross spontaneous DNA damage and mitotic aberrations. Furthermore, PALB2-deficiency disturbs three-dimensional spheroid morphology, increases the migrational capacity and invasiveness of the cells, and broadly alters their transcriptome profiles. TGFβ signaling and KRT14 expression are enhanced in PALB2-mutated cells and their inhibition and knock down, respectively, lead to partial restoration of cell functions. KRT14-positive cells are also more abundant with DNA damage than KRT14-negative cells. The obtained results indicate comprehensive cellular changes upon PALB2 mutations, even in the presence of half dosage of wild type PALB2 and demonstrate how PALB2 mutations may predispose their carriers to malignancy

Optical Genome Mapping Identifies a Second Xq27.1 Rearrangement Associated With Charcot-Marie-Tooth Neuropathy CMTX3

Abstract Background X-linked recessive type 3 Charcot–Marie–Tooth (CMTX3) is a rare subtype of childhood-onset CMT. To date, all reported CMTX3 patients share a common founder 78 kb insertion from chromosome 8 into the Xq27.1 palindrome region. Methods We conducted patient–parent trio optical genome mapping (OGM) on a male patient presenting with clinically diagnosed Dejerine–Sottas disease for whom initial standard diagnostic genetic tests, including whole-genome sequencing (WGS), yielded negative results. Results OGM analysis revealed a maternally inherited interchromosomal insertion from chromosome region 7q31.1 into Xq27.1. Coupled with manual reassessment of WGS data, this confirmed the molecular diagnosis of atypical CMTX3 and showed that the 122.4 kb inserted fragment contained DLD and partially LAMB1. Subsequent analyses confirmed that the rearrangement had arisen de novo in the proband's mother. Conclusion We report the second Xq27.1 rearrangement associated with CMTX3, providing novel clinical insights into its phenotypic and genotypic spectrum. Our findings highlight the importance of including genomic rearrangement analysis of Xq27.1 in standard diagnostic pipelines for childhood-onset CMT. Given the overlap in polyneuropathy phenotypes resulting from insertions from chromosomes 7 and 8 into the same Xq27.1 palindrome region, the pathogenic mechanism underlying peripheral neuropathy in CMTX3 likely involves dysregulation of genes within this region.Abstract Background X-linked recessive type 3 Charcot–Marie–Tooth (CMTX3) is a rare subtype of childhood-onset CMT. To date, all reported CMTX3 patients share a common founder 78 kb insertion from chromosome 8 into the Xq27.1 palindrome region. Methods We conducted patient–parent trio optical genome mapping (OGM) on a male patient presenting with clinically diagnosed Dejerine–Sottas disease for whom initial standard diagnostic genetic tests, including whole-genome sequencing (WGS), yielded negative results. Results OGM analysis revealed a maternally inherited interchromosomal insertion from chromosome region 7q31.1 into Xq27.1. Coupled with manual reassessment of WGS data, this confirmed the molecular diagnosis of atypical CMTX3 and showed that the 122.4 kb inserted fragment contained DLD and partially LAMB1. Subsequent analyses confirmed that the rearrangement had arisen de novo in the proband's mother. Conclusion We report the second Xq27.1 rearrangement associated with CMTX3, providing novel clinical insights into its phenotypic and genotypic spectrum. Our findings highlight the importance of including genomic rearrangement analysis of Xq27.1 in standard diagnostic pipelines for childhood-onset CMT. Given the overlap in polyneuropathy phenotypes resulting from insertions from chromosomes 7 and 8 into the same Xq27.1 palindrome region, the pathogenic mechanism underlying peripheral neuropathy in CMTX3 likely involves dysregulation of genes within this region

Case-control analysis of truncating mutations in DNA damage response genes connects TEX15 and FANCD2 with hereditary breast cancer susceptibility

Several known breast cancer susceptibility genes encode proteins involved in DNA damage response (DDR) and are characterized by rare loss-of-function mutations. However, these explain less than half of the familial cases. To identify novel susceptibility factors, 39 rare truncating mutations, identified in 189 Northern Finnish hereditary breast cancer patients in parallel sequencing of 796 DDR genes, were studied for disease association. Mutation screening was performed for Northern Finnish breast cancer cases (n = 578-1565) and controls (n = 337-1228). Mutations showing potential cancer association were analyzed in additional Finnish cohorts.c.7253dupT in TEX15, encoding a DDR factor important in meiosis, associated with hereditary breast cancer (p = 0.018) and likely represents a Northern Finnish founder mutation. A deleterious c.2715 + 1G > A mutation in the Fanconi anemia gene, FANCD2, was over two times more common in the combined Finnish hereditary cohort compared to controls. A deletion (c.640_644del5) in RNF168, causative for recessive RIDDLE syndrome, had high prevalence in majority of the analyzed cohorts, but did not associate with breast cancer. In conclusion, truncating variants in TEX15 and FANCD2 are potential breast cancer risk factors, warranting further investigations in other populations. Furthermore, high frequency of RNF168 c.640_644del5 indicates the need for its testing in Finnish patients with RIDDLE syndrome symptoms.Peer reviewe

ATM c.7570G>C is a high-risk allele for breast cancer

ATM is generally described as a moderate-risk breast cancer susceptibility gene. However, some of ATM variants might encounter higher risk. ATM c.7570G>C, p.Ala2524Pro, (rs769142993) is a pathogenic Finnish founder variant causative for recessively inherited ataxia-telangiectasia. At cellular level, it has been reported to have a dominant-negative effect. ATM c.7570G>C has recurrently been described in Finnish breast cancer families and unselected case cohorts collected from different parts of the country, but the rarity of the allele (MAF 0.0002772 in Finns) and lack of confirming segregation analyses have prevented any conclusive risk estimates. Here, we describe seven families from genetic counseling units with ATM c.7570G>C variant showing co-segregation with breast cancer. Further analysis of the unselected breast cancer cohort from Northern Finland (n = 1822), a geographical region previously indicated to have enrichment of the variant, demonstrated that c.7570G>C significantly associates with breast cancer, and the risk is estimated as high (odds ratio [OR] = 8.5, 95% confidence interval [CI] = 1.04-62.46, P = .018). Altogether, these results place ATM c.7570G>C variant among the high-risk alleles for breast cancer, which should be taken into consideration in genetic counseling