# US 2021/0241850 A1 · Fragile X Syndrome AGG Interruption Genotyping > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0241850 A1 - Document ID: US20210241850A1 - Status: Published application - Kind: A1 - Application number: 17/162,120 - Filed: 2021-01-29 - Published: 2021-08-05 - Earliest claimed: 2020-01-30 - Assignees: Laboratory Corporation of America Holdings - Applicants: Laboratory Corporation of America Holdings - Inventors: Zhenxi Zhang; Matt Robinson; Patricia Okamoto - Web page: https://patentgrep.com/patent/US20210241850A1 ## CPC - G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection - G16B 40/10: Signal processing, e.g. from mass spectrometry [MS] or from PCR - C12Q 1/686: Polymerase chain reaction [PCR] - G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present disclosure relates to Fragile X Syndrome (FXS) clinical testing, and in particular to a FXS AGG interruption polymerase chain reaction (PCR) assay and an AGG interruption genotyping algorithm for implementation into clinical testing. Particularly, aspects are directed to obtaining raw data from the FXS assay performed on a sample, iteratively searching the raw data and identifying one or more AGG peaks on the first allele using a first set of search spaces determined based on an expected AGG peak size, determining a number of CGG repeats downstream of a final AGG interruption and a number of CGG repeats preceding a first AGG interruption on the first allele based on the one or more AGG peaks, and generating a AGG genotype for the first allele based on the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption. ## Claims ### Claim 1 A method comprising: obtaining raw data from a Fragile X Syndrome (FXS) assay performed on a sample using a genetic analyzer, wherein the raw data comprises gene-specific (GS) polymerase chain reaction (PCR) data and AGG interruption PCR data resolved by capillary electrophoresis; determining an expected AGG peak size for a first allele identified in the raw data; iteratively searching the raw data and identifying one or more AGG peaks on the first allele using a first set of search spaces determined based on the expected AGG peak size; determining a number of CGG repeats downstream of a final AGG interruption on the first allele based on the GS PCR data and the one or more AGG peaks identified on the first allele; determining a number of CGG repeats preceding a first AGG interruption on the first allele based on the GS PCR data and the one or more AGG peaks identified on the first allele; generating a AGG genotype for the first allele based on the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption; and providing the AGG genotype for the first allele. ### Claim 2 (depends on 1) The method of claim 1, further comprising determining a number of CGG repeats separated by any two neighboring AGG interruptions on the first allele based on the GS PCR data and the one or more AGG peaks identified on the first allele, wherein the AGG genotype for the first allele is generated based on the number of CGG repeats downstream of the final AGG interruption, the number of CGG repeats separated by any two neighboring AGG interruptions, and the number of CGG repeats preceding the first AGG interruption. ### Claim 3 (depends on 2) The method of claim 2, further comprising iteratively searching the raw data and identifying one or more AGG peaks on a second allele using a second set of search spaces determined based on the expected AGG peak size; determining a number of CGG repeats downstream of a final AGG interruption on the second allele based on the GS PCR data and the one or more AGG peaks identified on the second allele; determining a number of CGG repeats preceding a first AGG interruption on the second allele based on the GS PCR data and the one or more AGG peaks identified on the second allele; generating a AGG genotype for the second allele based on the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption; and providing the AGG genotype for the second allele, wherein: (i) the first allele is a normal allele and the second allele is a premutation allele, (ii) the first allele is a normal allele and the second allele is a different normal allele, or (iii) the first allele is a premutation allele and the second allele is a different premutation allele. ### Claim 4 (depends on 3) The method of claim 3, further comprising determining a risk score for a subject associated with the sample based on the AGG genotype generated for the first allele, the second allele, or both the first allele and the second allele, wherein the risk score identifies a risk of the subject developing late-onset neurodegenerative disease fragile X-associated tremor/ataxia syndrome (FXTAS) or fragile X-associated primary ovarian insufficiency (FXPOI) or transmitting a full mutation allele to their offspring or any combination thereof. ### Claim 5 (depends on 3) The method of claim 3, wherein the iteratively searching the raw data and identifying one or more AGG peaks on the first allele comprises: determining a first search space of the first set of search spaces for the first allele based on the expected AGG peak size; searching the raw data and identifying an initial AGG peak on the first allele using a first search space; determining a second search space for the first allele based on a peak size of the initial AGG peak on the first allele; and iteratively searching the raw data and identifying one or more additional AGG peaks on the first allele using the second search space, wherein the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption are determined for the first allele based on the GS PCR data, the initial peak identified on the first allele, and the one or more additional peaks identified on the first allele. ### Claim 6 (depends on 5) The method of claim 5, wherein the iteratively searching the raw data and identifying one or more AGG peaks on the second allele comprises: determining a first search space of the second set of search spaces for the second allele based on the expected AGG peak size; searching the raw data and identifying an initial AGG peak on the second allele using a second search space; determining a second search space for the second allele based on a peak size of the initial AGG peak on the second allele; and iteratively searching the raw data and identifying one or more additional AGG peaks on the second allele using the second search space, wherein the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption are determined for the second allele based on the GS PCR data, the initial peak identified on the second allele, and the one or more additional peaks identified on the second allele. ### Claim 7 (depends on 5) The method of claim 5, wherein once the one or more AGG peaks on the first allele are identified, the one or more AGG peaks on the first allele are removed from the raw data prior to iteratively searching the raw data and identifying the one or more AGG peaks on the second allele. ### Claim 8 A system comprising: one or more data processors; and a non-transitory computer readable storage medium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform actions including: obtaining raw data from a Fragile X Syndrome (FXS) assay performed on a sample using a genetic analyzer, wherein the raw data comprises gene-specific (GS) polymerase chase reaction (PCR) data and AGG interruption PCR data resolved by capillary electrophoresis; determining an expected AGG peak size for a first allele identified in the raw data; iteratively searching the raw data and identifying one or more AGG peaks on the first allele using a first set of search spaces determined based on the expected AGG peak size; determining a number of CGG repeats downstream of a final AGG interruption on the first allele based on the GS PCR data and the one or more AGG peaks identified on the first allele; determining a number of CGG repeats preceding a first AGG interruption on the first allele based on the GS PCR data and the one or more AGG peaks identified on the first allele; generating a AGG genotype for the first allele based on the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption; and providing the AGG genotype for the first allele. ### Claim 9 (depends on 8) The system of claim 8, wherein the actions further include determining a number of CGG repeats separated by any two neighboring AGG interruptions on the first allele based on the GS PCR data and the one or more AGG peaks identified on the first allele, wherein the AGG genotype for the first allele is generated based on the number of CGG repeats downstream of the final AGG interruption, the number of CGG repeats separated by any two neighboring AGG interruptions, and the number of CGG repeats preceding the first AGG interruption. ### Claim 10 (depends on 9) The system of claim 9, wherein the actions further include: iteratively searching the raw data and identifying one or more AGG peaks on a second allele using a second set of search spaces determined based on the expected AGG peak size; determining a number of CGG repeats downstream of a final AGG interruption on the second allele based on the GS PCR data and the one or more AGG peaks identified on the second allele; determining a number of CGG repeats preceding a first AGG interruption on the second allele based on the GS PCR data and the one or more AGG peaks identified on the second allele; generating a AGG genotype for the second allele based on the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption; and providing the AGG genotype for the second allele, wherein: (i) the first allele is a normal allele and the second allele is a premutation allele, (ii) the first allele is a normal allele and the second allele is a different normal allele, or (iii) the first allele is a premutation allele and the second allele is a different premutation allele. ### Claim 11 (depends on 10) The system of claim 10, wherein the actions further include determining a risk score for a subject associated with the sample based on the AGG genotype generated for the first allele, the second allele, or both the first allele and the second allele, and wherein the risk score identifies a risk of the subject developing late-onset neurodegenerative disease fragile X-associated tremor/ataxia syndrome (FXTAS) or fragile X-associated primary ovarian insufficiency (FXPOI) or transmitting a full mutation allele to their offspring or any combination thereof. ### Claim 12 (depends on 10) The system of claim 10, wherein the iteratively searching the raw data and identifying one or more AGG peaks on the first allele comprises: determining a first search space of the first set of search spaces for the first allele based on the expected AGG peak size; searching the raw data and identifying an initial AGG peak on the first allele using a first search space; determining a second search space for the first allele based on a peak size of the initial AGG peak on the first allele; and iteratively searching the raw data and identifying one or more additional AGG peaks on the first allele using the second search space, wherein the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption are determined for the first allele based on the GS PCR data, the initial peak identified on the first allele, and the one or more additional peaks identified on the first allele. ### Claim 13 (depends on 12) The system of claim 12, wherein the iteratively searching the raw data and identifying one or more AGG peaks on the second allele comprises: determining a first search space of the second set of search spaces for the second allele based on the expected AGG peak size; searching the raw data and identifying an initial AGG peak on the second allele using a second search space; determining a second search space for the second allele based on a peak size of the initial AGG peak on the second allele; and iteratively searching the raw data and identifying one or more additional AGG peaks on the second allele using the second search space, wherein the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption are determined for the second allele based on the GS PCR data, the initial peak identified on the second allele, and the one or more additional peaks identified on the second allele. ### Claim 14 (depends on 12) The system of claim 12, wherein once the one or more AGG peaks on the first allele are identified, the one or more AGG peaks on the first allele are removed from the raw data prior to iteratively searching the raw data and identifying the one or more AGG peaks on the second allele. ### Claim 15 A computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform actions including: obtaining raw data from a Fragile X Syndrome (FXS) assay performed on a sample using a genetic analyzer, wherein the raw data comprises gene-specific (GS) polymerase chase reaction (PCR) data and AGG interruption PCR data resolved by capillary electrophoresis; determining an expected AGG peak size for a first allele identified in the raw data; iteratively searching the raw data and identifying one or more AGG peaks on the first allele using a first set of search spaces determined based on the expected AGG peak size; determining a number of CGG repeats downstream of a final AGG interruption on the first allele based on the GS PCR data and the one or more AGG peaks identified on the first allele; determining a number of CGG repeats preceding a first AGG interruption on the first allele based on the GS PCR data and the one or more AGG peaks identified on the first allele; generating a AGG genotype for the first allele based on the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption; and providing the AGG genotype for the first allele. ### Claim 16 (depends on 15) The computer-program product of claim 15, wherein the actions further include determining a number of CGG repeats separated by any two neighboring AGG interruptions on the first allele based on the GS PCR data and the one or more AGG peaks identified on the first allele, wherein the AGG genotype for the first allele is generated based on the number of CGG repeats downstream of the final AGG interruption, the number of CGG repeats separated by any two neighboring AGG interruptions, and the number of CGG repeats preceding the first AGG interruption. ### Claim 17 (depends on 16) The computer-program product of claim 16, wherein the actions further include: iteratively searching the raw data and identifying one or more AGG peaks on a second allele using a second set of search spaces determined based on the expected AGG peak size; determining a number of CGG repeats downstream of a final AGG interruption on the second allele based on the GS PCR data and the one or more AGG peaks identified on the second allele; determining a number of CGG repeats preceding a first AGG interruption on the second allele based on the GS PCR data and the one or more AGG peaks identified on the second allele; generating a AGG genotype for the second allele based on the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption; and providing the AGG genotype for the second allele, wherein: (i) the first allele is a normal allele and the second allele is a premutation allele, (ii) the first allele is a normal allele and the second allele is a different normal allele, or (iii) the first allele is a premutation allele and the second allele is a different premutation allele. ### Claim 18 (depends on 17) The computer-program product of claim 17, wherein the actions further include determining a risk score for a subject associated with the sample based on the AGG genotype generated for the first allele, the second allele, or both the first allele and the second allele, and wherein the risk score identifies a risk of the subject developing late-onset neurodegenerative disease fragile X-associated tremor/ataxia syndrome (FXTAS) or fragile X-associated primary ovarian insufficiency (FXPOI) or transmitting a full mutation allele to their offspring or any combination thereof. ### Claim 19 (depends on 17) The computer-program product of claim 17, wherein the iteratively searching the raw data and identifying one or more AGG peaks on the first allele comprises: determining a first search space of the first set of search spaces for the first allele based on the expected AGG peak size; searching the raw data and identifying an initial AGG peak on the first allele using a first search space; determining a second search space for the first allele based on a peak size of the initial AGG peak on the first allele; and iteratively searching the raw data and identifying one or more additional AGG peaks on the first allele using the second search space, wherein the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption are determined for the first allele based on the GS PCR data, the initial peak identified on the first allele, and the one or more additional peaks identified on the first allele. ### Claim 20 (depends on 19) The computer-program product of claim 19, wherein the iteratively searching the raw data and identifying one or more AGG peaks on the second allele comprises: determining a first search space of the second set of search spaces for the second allele based on the expected AGG peak size; searching the raw data and identifying an initial AGG peak on the second allele using a second search space; determining a second search space for the second allele based on a peak size of the initial AGG peak on the second allele; and iteratively searching the raw data and identifying one or more additional AGG peaks on the second allele using the second search space, wherein the number of CGG repeats downstream of the final AGG interruption and the number of CGG repeats preceding the first AGG interruption are determined for the second allele based on the GS PCR data, the initial peak identified on the second allele, and the one or more additional peaks identified on the second allele.