# US 10,870,886 B2 · Molecular diagnosis of FSHD by epigenetic signature > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,870,886 B2 - Document ID: US10870886B2 - Status: Granted - Kind: B2 - Application number: 15/516,291 - Filed: 2015-10-07 - Published: 2017-10-26 - Granted: 2020-12-22 - Earliest claimed: 2014-10-09 - Assignees: University of Massachusetts Medical Center - Applicants: University of Massachusetts Medical Center - Inventors: Peter L. Jones; Takako Jones; Johnny Salameh; Colin Quinn; Oliver D. King - Web page: https://patentgrep.com/patent/US10870886B2 ## CPC - C12Q 1/6883: for diseases caused by alterations of genetic material - C12Q 2600/118: Prognosis of disease development - C12Q 2600/154: Methylation markers - 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 invention includes methods of determining whether an individual in need thereof has, or is at risk of developing, facioscapulohumeral muscular dystrophy (FSHD). ## Claims ### Claim 1 A method of determining whether a human subject has, or is at risk of developing, facioscapulohumeral muscular dystrophy 2 (FSHD2) comprising performing a DNA methylation analysis of deoxycytosine-phosphate-deoxyguanine dinucleotides (CpGs) in a distal D4Z4 repeat unit of a D4Z4 repeat array and a proximal region of an A-type subtelomere of each of a chromosome 4qA allele and a chromosome 4qA-L allele of chromosome 4q, if present, wherein the DNA methylation analysis comprises: i) performing polymerase chain reaction (PCR) using genomic DNA from at least one sample from the subject and a forward primer comprising the sequence of SEQ ID NO: 8 and a first reverse primer comprising the sequence of SEQ ID NO: 9 to obtain at least one first 4qA PCR product if the genomic DNA comprises a 4qA allele; ii) performing PCR using the at least one first 4qA PCR product, the forward primer comprising the sequence of SEQ ID NO: 8 and a second reverse primer comprising the sequence of SEQ ID NO: 10 to obtain at least one second 4qA PCR product if the genomic DNA comprises a 4qA allele; iii) performing PCR using genomic DNA from at least one sample from the subject and a forward primer comprising the sequence of SEQ ID NO: 11 and the first reverse primer comprising the sequence of SEQ ID NO: 9 to obtain at least one first 4qA-L PCR product if the genomic DNA comprises a 4qA-L allele; iv) performing PCR using the at least one first 4qA-L PCR product, the forward primer comprising the sequence of SEQ ID NO: 11 and the second reverse primer comprising the sequence of SEQ ID NO: 10 to obtain at least one second 4qA-L PCR product if the genomic DNA comprises a 4qA-L allele; v) performing bisulfite sequencing for each of the at least one second 4qA PCR product and the at least one second 4qA-L PCR product, if the genomic DNA comprises a 4qA allele and/or 4qA-L allele, respectively; vi) quantifying methylation of the CpGs in the distal D4Z4 repeat unit and the proximal region of the A-type subtelomere in each of the at least one second 4qA PCR product and the at least one second 4qA-L PCR product to obtain the first quartile of the methylation percent of the distal D4Z4 repeat unit and the proximal region of the A-type subtelomere of each of the second 4qA PCR product and the second 4qA-L PCR product, wherein if 25% or less of the CpGs in the distal D4Z4 repeat unit and the proximal region of the A-type subtelomere in the first quartile of the second 4qA PCR product are methylated or if 25% or less of the CpGs in the first quartile of the second 4qA-L PCR product are methylated, then the subject has, or is at risk of developing, FSHD2. ### Claim 2 (depends on 1) The method of claim 1 wherein if between about 35% of the CpGs in the first quartile of the analysis of the distal D4Z4 repeat unit and the proximal region of the A-type subtelomere are methylated, and the individual exhibits no detectable muscle weakness, then the individual is diagnosed as an FSHD2 carrier. ### Claim 3 (depends on 1) The method of claim 1 wherein if greater than about 35% of the CpGs in the first quartile of the analysis of the distal D4Z4 repeat unit and the proximal region of the A-type subtelomere are methylated, then the individual does not have, or is not at risk of developing, FSHD2. ### Claim 4 (depends on 1) The method of claim 1 wherein if no at least one first 4qA PCR product and no at least one first 4qA-L PCR product are produced by the PCR, then the subject does not have, or is not at risk of developing, FSHD2. ### Claim 5 (depends on 1) The method of claim 1, further comprising determining whether the distal D4Z4 repeat of the D4Z4 repeat array on chromosome 4q unit includes CpG #55, wherein if analysis of the distal D4Z4 repeat unit results in CpG #55 being absent, relative to a reference sequence, from all sequences of the at least one second 4qA PCR product and the at least one second 4qA-L PCR product being analyzed, then the subject does not have, or is not at risk of developing, FSHD2. ### Claim 6 (depends on 1) The method of claim 1, further comprising determining whether the distal D4Z4 repeat of the D4Z4 repeat array on chromosome 4q unit includes CpG #16 and CpG #55, wherein if analysis of the distal D4Z4 repeat unit results in both CpG #16 and CpG #55 being absent, relative to a reference sequence, from all sequences of the at least one second 4qA PCR product and the at least one second 4qA-L PCR product being analyzed, then the subject does not have, or is not at risk of developing, FSHD2. ### Claim 7 (depends on 1) The method of claim 1 wherein the individual is symptomatic for FSHD but produces a negative genetic test for FSHD1. ### Claim 8 (depends on 2) The method of claim 2 wherein the individual is an asymptomatic carrier of FSHD2. ### Claim 9 (depends on 1) The method of claim 1 wherein the methylation analysis is performed using bisulfate converted genomic DNA. ### Claim 10 (depends on 1) The method of claim 1 further comprising determining the sequence of a subtelomere of chromosome 4q, a subtelomere of 10q, one or more D4Z4 repeat units or a combination thereof. ### Claim 11 (depends on 1) The method of claim 1 wherein the sequence is determined by performing Sanger sequencing or next generation sequencing. ### Claim 12 (depends on 1) The method of claim 1 wherein the sample comprises one or more samples from one or more subjects that do not have FSHD. ### Claim 13 (depends on 1) The method of claim 1 wherein the sample is one or more fluids, one or more tissues, one or more cells or a combination thereof obtained from the subject. ### Claim 14 (depends on 13) The method of claim 13 wherein the sample is saliva, blood, myocytes, fibroblasts, tissue, hair or cultured cells.