Periodic Reporting for period 1 - RESEMBLE (Long-read sequencing to resolve the missing heritability in patients suspected of PTEN hamartoma tumour syndrome)
Reporting period: 2022-05-15 to 2024-05-14
Individuals with a genetic tumour risk syndrome are at high risk to develop cancer. One of these syndromes is PTEN hamartoma tumour syndrome (PHTS). PHTS is associated with pathogenic variants (PVs) in the PTEN gene. Unfortunately, in the majority of patients suspected of PHTS no PV in PTEN is identified. These patients are considered PHTS-like. The low diagnosis rate in this group of patients suggests the possibility of PVs in regions of PTEN that are not covered in routine diagnostics or suggests that PVs in other genes in the PI3K/AKT/mTOR pathway result in PHTS-like disease. The current state-of-the-art techniques used to detect genetic variants in clinical diagnostics are mainly based on short- read next generation sequencing (NGS). The introduction of short-read NGS technologies into genomic testing pipelines has led to a vast increase in diagnoses and many novel disease-gene associations. Nevertheless, we are still unable to detect complex genetic rearrangements and variants in less accessible regions of the genome, such as promoters and intronic regions, which are more likely to be GC rich and/or harbour repetitive sequences hampering short-read NGS. These limitations can be overcome with the use of newly developed long-read sequencing chemistries, which goes beyond the current state-of-the-art, and allows for more accurate sequencing of structural variants and increased coverage of difficult regions in the genome. This advanced sequencing technology can also be applied in a targeted manner, when there is good phenotypic evidence to suspect alterations in specific genes or genomic regions, or genome-wide to identify new risk loci. Long-read sequencing technologies have shown potential to improve the diagnostic yield of other diseases, such as polycystic kidney disease and intellectual disability. However, this has never been done in PHTS-like patients. Therefore, I aim to develop a long-read sequencing (LRS) approach for PHTS-like patients, which will go beyond the currently state-of-the-art short-read sequencing approaches. I will:
- develop a PTEN-targeted LRS assay;
- perform this targeted assay on a highly selective cohort of PHTS-like families;
- perform whole genome LRS on families that were not solved by targeted LRS to identify novel gene-disease associations;
- Functionally validate potential findings.
Via this work, I aim to improve the diagnostic yield for PHTS by identifying novel genetic mechanisms and genes underlying disease in PHTS-like patients. The unique interdisciplinary structure of the Department of Human Genetics at Radboudumc will allow to rapidly implement genetic testing for the newly identified genetic causes for PHTS. Identification of the precise underlying cause of PHTS will enable cascade genetic testing of at-risk family members to determine their status for the causal variant and thus, risk of disease to them and any future off-spring. Furthermore, this approach will allow the implementation of preventive care strategies for the family members at risk. Overall, my proposal will facilitate a personalized care approach for PHTS(-like) patients and their families for improved clinical outcomes and reduced disease morbidity.
- develop a PTEN-targeted LRS assay;
- perform this targeted assay on a highly selective cohort of PHTS-like families;
- perform whole genome LRS on families that were not solved by targeted LRS to identify novel gene-disease associations;
- Functionally validate potential findings.
Via this work, I aim to improve the diagnostic yield for PHTS by identifying novel genetic mechanisms and genes underlying disease in PHTS-like patients. The unique interdisciplinary structure of the Department of Human Genetics at Radboudumc will allow to rapidly implement genetic testing for the newly identified genetic causes for PHTS. Identification of the precise underlying cause of PHTS will enable cascade genetic testing of at-risk family members to determine their status for the causal variant and thus, risk of disease to them and any future off-spring. Furthermore, this approach will allow the implementation of preventive care strategies for the family members at risk. Overall, my proposal will facilitate a personalized care approach for PHTS(-like) patients and their families for improved clinical outcomes and reduced disease morbidity.
Patient cohort
The PTHS-like cohort included patients who were genetically counselled for PHTS and who tested negative for gPVs in the coding regions of PTEN. Patients were included if they were ≥18 years and were diagnosed with: i) ≥2 major PHTS features (breast-, endometrial-, thyroid cancer, macrocephaly, hamartomas, ganglioneuromas, trichilemmomas); ii) 1 major feature and ≥2 minor PHTS features (colorectal-, renal cancer, benign breast disease, benign thyroid disease, lipomas, vascular malformations); iii) 1 non-cancer major feature; or iv) any PHTS-associated cancer at age ≤35 years. All pathology-related features were extracted from the Dutch Nationwide Pathology Databank (PALGA).
Of 281 individuals of that were tested negative for PTEN, A total of 62 PHTS-like patients met the inclusion criteria and were successfully sequenced. From this cohort 37 individuals were diagnosed with breast cancer; six with endometrial cancer; sixteen with thyroid cancer; three with renal cancer and six with colorectal cancer. Twenty-three individuals have macrocephaly and eight individuals were diagnosed with Lhermitte Duclos (n=2), hamartomas (n = 2), ganglioneuromas (n = 2) or trichilemmomas (n = 2).
Targeted long-read sequencing of PTEN
In order to apply a T-LRS approach for PTEN, Long-range PCR amplicons sizing between ~8-16kb with a 1 kb overlap were designed to cover the full length of PTEN (108.31 kb). This targeted amplicon-based approach was chosen as it can also be applied on older DNA samples from PHTS-like patients. The design of the T-LRS approach for PTEN was successful, using a total of nine amplicons to capture the region of interest. The T-LRS was performed on a Pacific Biosciences Sequel II sequencer system. On average, each sample was sequenced at a median read depth of 254 reads [median range: 156-373 reads per sample]. After variant prioritization and in silico predictions for pathogenicity, two variants of interest were identified in two individuals in the 5’UTR and 3’UTR of PTEN.
T-LRS in PHTS-like patients allows for identification of variants in non-coding regions within PTEN. Functional analysis of these variants of interest will be carried out in order to determine their effect on expression of PTEN. Upon confirmation of the pathogenicity of these variants, this approach may lead to a genetic diagnosis for 3% of the tested PHTS-like patients.
Whole genome long-read sequencing
In addition to the T-LRS, eight individuals were selected for whole genome LRS. These individuals were also tested with T-LRS but remained negative for any putative causative variants. Whole genome LRS was performed on a Pacific Biosciences Revio sequencer system. So far, no variants have been identified that explain the phenotype of the patients.
Functional validation of variants in PTEN
In addition to the follow-up of variants that were identified through T-LRS, variants of uncertain significance (VUSs) in PTEN were also assessed for functional follow-up. These VUSs were previously identified in an ERN-GENTURIS PHTS cohort (n=510). All variants were classified as a VUS using the PTEN-specific ACMG guidelines. In order for these variants to be eligible for functional follow-up they needed to have a spliceAI score of >0.1 with no published functional data to date. Subsequently, these variants were then considered for a splice assay. Amplicons spanning one or two PTEN exons that harboured the identified VUSs were designed and cloned into a minigene vector using gateway cloning. Site-directed mutagenesis was used to generate the variants of interest and splice assays with mutant and wild type constructs were performed. Eight PTEN VUSs were identified for further functional testing. We identified five missense variants (c.77C>A, p.(Thr26Asn); c.89C>A, p.(Pro30Gln); c.94A>T, p.(Ile32Phe); c.801G>T, p.(Lys267Asn); c.929A>G, p.(Asp310Gly)) and three intronic variants (c.80-1G>C; c.165-18T>A; c.253+5G>C). These eight variants were distributed over six exon-intron regions, captured in five amplicons ranging between ~2.5-5.2 kb in size. The mini-gene splice assay has currently shown that variant c.253+5G>C results in vitro in altered splicing of PTEN.
The PTHS-like cohort included patients who were genetically counselled for PHTS and who tested negative for gPVs in the coding regions of PTEN. Patients were included if they were ≥18 years and were diagnosed with: i) ≥2 major PHTS features (breast-, endometrial-, thyroid cancer, macrocephaly, hamartomas, ganglioneuromas, trichilemmomas); ii) 1 major feature and ≥2 minor PHTS features (colorectal-, renal cancer, benign breast disease, benign thyroid disease, lipomas, vascular malformations); iii) 1 non-cancer major feature; or iv) any PHTS-associated cancer at age ≤35 years. All pathology-related features were extracted from the Dutch Nationwide Pathology Databank (PALGA).
Of 281 individuals of that were tested negative for PTEN, A total of 62 PHTS-like patients met the inclusion criteria and were successfully sequenced. From this cohort 37 individuals were diagnosed with breast cancer; six with endometrial cancer; sixteen with thyroid cancer; three with renal cancer and six with colorectal cancer. Twenty-three individuals have macrocephaly and eight individuals were diagnosed with Lhermitte Duclos (n=2), hamartomas (n = 2), ganglioneuromas (n = 2) or trichilemmomas (n = 2).
Targeted long-read sequencing of PTEN
In order to apply a T-LRS approach for PTEN, Long-range PCR amplicons sizing between ~8-16kb with a 1 kb overlap were designed to cover the full length of PTEN (108.31 kb). This targeted amplicon-based approach was chosen as it can also be applied on older DNA samples from PHTS-like patients. The design of the T-LRS approach for PTEN was successful, using a total of nine amplicons to capture the region of interest. The T-LRS was performed on a Pacific Biosciences Sequel II sequencer system. On average, each sample was sequenced at a median read depth of 254 reads [median range: 156-373 reads per sample]. After variant prioritization and in silico predictions for pathogenicity, two variants of interest were identified in two individuals in the 5’UTR and 3’UTR of PTEN.
T-LRS in PHTS-like patients allows for identification of variants in non-coding regions within PTEN. Functional analysis of these variants of interest will be carried out in order to determine their effect on expression of PTEN. Upon confirmation of the pathogenicity of these variants, this approach may lead to a genetic diagnosis for 3% of the tested PHTS-like patients.
Whole genome long-read sequencing
In addition to the T-LRS, eight individuals were selected for whole genome LRS. These individuals were also tested with T-LRS but remained negative for any putative causative variants. Whole genome LRS was performed on a Pacific Biosciences Revio sequencer system. So far, no variants have been identified that explain the phenotype of the patients.
Functional validation of variants in PTEN
In addition to the follow-up of variants that were identified through T-LRS, variants of uncertain significance (VUSs) in PTEN were also assessed for functional follow-up. These VUSs were previously identified in an ERN-GENTURIS PHTS cohort (n=510). All variants were classified as a VUS using the PTEN-specific ACMG guidelines. In order for these variants to be eligible for functional follow-up they needed to have a spliceAI score of >0.1 with no published functional data to date. Subsequently, these variants were then considered for a splice assay. Amplicons spanning one or two PTEN exons that harboured the identified VUSs were designed and cloned into a minigene vector using gateway cloning. Site-directed mutagenesis was used to generate the variants of interest and splice assays with mutant and wild type constructs were performed. Eight PTEN VUSs were identified for further functional testing. We identified five missense variants (c.77C>A, p.(Thr26Asn); c.89C>A, p.(Pro30Gln); c.94A>T, p.(Ile32Phe); c.801G>T, p.(Lys267Asn); c.929A>G, p.(Asp310Gly)) and three intronic variants (c.80-1G>C; c.165-18T>A; c.253+5G>C). These eight variants were distributed over six exon-intron regions, captured in five amplicons ranging between ~2.5-5.2 kb in size. The mini-gene splice assay has currently shown that variant c.253+5G>C results in vitro in altered splicing of PTEN.
In conclusion, we have been successful in designing and applying a T-LRS approach for PTEN. T-LRS in PHTS-like patients allows for identification of variants in non-coding regions within PTEN. Functional analysis of these variants of interest is carried out in order to determine their effect on expression of PTEN. Upon confirmation of the pathogenicity of these variants, this approach may lead to a genetic diagnosis for 3% of the tested PHTS-like patients.
In addition, upon final assessment, coding variants may also exert an effect on splicing and thus impact the function of PTEN. Splice effect analysis will help to further classify the pathogenicity of PTEN VUSs.
Overall, these findings will impact the lives of the affected patients by these variants as they now can receive a definitive genetic diagnosis and this will enable cascade genetic testing of at-risk family members through the clinical genetics service to determine their status for the causal variant and thus, risk of disease to them and any future off-spring. Furthermore, this will allow the implementation of preventative care strategies for the family members at risk, such as regular surveillance for breast-, thyroid-, renal-, colorectal cancer and melanoma, as recommended in the clinical guideline for PHTS. Overall, this will facilitate a personalized care approach of PHTS patients and their families for improved clinical outcomes and reduced disease morbidity.
In addition, upon final assessment, coding variants may also exert an effect on splicing and thus impact the function of PTEN. Splice effect analysis will help to further classify the pathogenicity of PTEN VUSs.
Overall, these findings will impact the lives of the affected patients by these variants as they now can receive a definitive genetic diagnosis and this will enable cascade genetic testing of at-risk family members through the clinical genetics service to determine their status for the causal variant and thus, risk of disease to them and any future off-spring. Furthermore, this will allow the implementation of preventative care strategies for the family members at risk, such as regular surveillance for breast-, thyroid-, renal-, colorectal cancer and melanoma, as recommended in the clinical guideline for PHTS. Overall, this will facilitate a personalized care approach of PHTS patients and their families for improved clinical outcomes and reduced disease morbidity.