Methods to detect a silent carrier genotype

Abstract

Provided herein are methods and compositions for the detection of silent carriers of chromosomal deletion alleles in a human subject using haploid cells (e.g., sperm cells or egg cells) derived from the subject. The methods provided herein allow for the detection of silent (2+0) carriers of SMA, where the individual has a deletion of the SMN1 gene on one chromosome 5 homolog and two or more copies of the SMN1 gene on the other chromosome 5 homolog.

1 .- 33 . (canceled) 
     
     
         34 . A kit comprising:
 (a) a pair of oligonucleotide primers for amplification of a target region of the SMN1 gene, wherein the target region is deleted in target gene null allele of an SMN1 silent carrier, and   (b) a pair of oligonucleotide primers for amplification of a target region of a reference gene that is not deleted in an SMN1 silent carrier; and   (c) one or more reagents for performing a nucleic acid amplification reaction.   
     
     
         35 . The kit of  claim 34 , wherein the kit comprises nucleotide triphosphates, a thermostable polymerase, and/or a suitable buffer. 
     
     
         36 . The kit of  claim 34 , wherein the reference gene is selected from among CFTR, GAPDH, HMBS, B2M, HPRT1, RPL13A, SDHA, TBP, UBC, YWHAZ, PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2, and ATP5O. 
     
     
         37 . The kit of  claim 34 , wherein the target gene amplification product comprises exon 7 of SMN1 or a portion thereof. 
     
     
         38 . A method for identifying a subject as a silent carrier of a target gene null allele comprising:
 (a) performing a plurality of quantitative nucleic acid amplification reactions, wherein each nucleic acid amplification reaction comprises
 (i) a genomic DNA sample obtained from a single haploid cell from the subject, 
 (ii) at least one pair of oligonucleotide primers for amplification of a target region of a target gene, wherein the target region is absent in a target gene null allele, and 
 (iii) at least one pair of oligonucleotide primers for amplification of a target region of a reference gene; 
   (b) detecting the presence or absence of the target gene amplification product;   (c) detecting the presence or absence of the reference gene amplification product;   (d) determining a ratio of detected target gene amplification product to detected reference gene amplification product; and   (e) characterizing the subject as a silent carrier of the target gene null allele if the ratio of target gene amplification products to reference gene amplification products is at or below a threshold level between about 0.5 and about 0.8.   
     
     
         39 . A method for identifying a subject as a silent carrier of a target gene null allele comprising:
 (a) performing a plurality of nucleic acid amplification reactions, wherein each nucleic acid amplification reaction comprises
 (i) a genomic DNA sample obtained from a subject suspected of being a silent carrier of a target gene null allele; 
 (ii) at least one pair of oligonucleotide primers for amplification of a target region of a target gene, wherein the target region is absent in the target gene null allele, and wherein an oligonucleotide primer of the pair of oligonucleotide primers comprises a unique barcode; and 
 (iii) at least one pair of oligonucleotide primers for amplification of a target region of a reference gene; 
   (b) detecting the presence or absence of a target gene amplification product;   (c) detecting the presence or absence of a reference gene amplification product;   (d) determining a ratio of detected target gene amplification product to detected reference gene amplification product; and   (e) characterizing the subject as a silent carrier of the target gene null allele based on the ratio of target gene amplification product to reference gene amplification product.   
     
     
         40 . A method of preparing a sample from a subject suspected of being a silent carrier of a target gene null allele comprising:
 (a) performing a plurality of nucleic acid amplification reactions, wherein each nucleic acid amplification reaction comprises
 (i) a genomic DNA sample obtained from a subject suspected of being a silent carrier of a target gene null allele; 
 (ii) at least one pair of oligonucleotide primers for amplification of a target region of a target gene, wherein the target region is absent in the target gene null allele, and wherein an oligonucleotide primer of the pair of oligonucleotide primers comprises a unique barcode; and 
 (iii) at least one pair of oligonucleotide primers for amplification of a target region of a reference gene; 
   (b) detecting the presence or absence of a target gene amplification product;   (c) detecting the presence or absence of a reference gene amplification product;   (d) determining a ratio of detected target gene amplification product to detected reference gene amplification product; and   (e) characterizing the subject as a silent carrier of the target gene null allele based on the ratio of target gene amplification product to reference gene amplification product.   
     
     
         41 . The method of  claim 40 , wherein the subject is suspected of having a deletion of the SMN1 gene on one chromosome 5 homolog and two or more copies of the SMN1 gene on the other chromosome 5 homolog. 
     
     
         42 . The method of  claim 40 , wherein detecting the presence or absence of the target gene amplification product and reference gene amplification product comprises sequencing the target gene amplification product and reference gene amplification product. 
     
     
         43 . The method of  claim 42 , wherein sequencing comprises bulk sequencing, next generation sequence, or massively parallel sequencing. 
     
     
         44 . The method of  claim 40 , wherein genomic DNA sample is obtained from a haploid cell. 
     
     
         45 . The method of  claim 40 , wherein the genomic DNA sample is obtained from a diploid cell. 
     
     
         46 . The method of  claim 45 , wherein the diploid cell is a blood cell. 
     
     
         47 . The method of  claim 40 , wherein the reference gene is selected from among CFTR, GAPDH, HMBS, B2M, HPRT1, RPL13A, SDHA, TBP, UBC, YWHAZ, PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2, and ATP50. 
     
     
         48 . The method of  claim 40 , wherein the target gene is SMN1. 
     
     
         49 . The method of  claim 40 , wherein the target gene amplification product comprises exon 7 of SMN1 or a portion thereof.