Noninvasive prenatal genotyping of fetal sex chromosomes

Abstract

Methods, apparatuses, and system are provided for analyzing a maternal sample to determine whether a male fetus of a pregnant female has inherited an X-linked mutation from the mother. A percentage of fetal DNA in the sample is obtained, and cutoff values for the two possibilities (fetus inherits mutant or normal allele) are determined. A proportion of mutant alleles relative to a normal allele on the X-chromosome can then be compared to the cutoff values to make a classification of which allele is inherited. Alternatively, a number of alleles from a target region on the X-chromosome can be compared to a number of alleles from a reference region on the X-chromosome to identify a deletion or amplification. The fetal DNA percentage can be computed by counting reactions with a fetal-specific allele, and correcting the number to account for a statistical distribution among the reactions.

What is claimed is: 
     
         1 . A method for determining whether a male fetus of a pregnant female has an X-linked mutation, wherein the pregnant female is heterozygous for a mutant and a normal allele at a locus on the X chromosome, the method comprising:
 receiving, by a computer system, data from a plurality of reactions, each involving one or more nucleic acid molecules from a biological sample, the biological sample including cell-free nucleic acid molecules from the pregnant female and from the male fetus,   wherein the data includes:
 a first set of quantitative data indicating a first amount of the mutant allele at the locus; and 
 a second set of quantitative data indicating a second amount of the normal allele at the locus; 
   determining, by the computer system, a parameter from the first amount and the second amount, wherein the parameter represents a relative amount between the first and second amounts;   obtaining, by the computer system, a percentage Pf of fetal nucleic acid molecules in the biological sample;   calculating, by the computer system, a first cutoff value for determining whether the fetus has inherited the mutant allele at the locus, wherein the first cutoff value is derived at least from a first proportion of k/(1+k−Pf), where k is a number of mutant alleles on a mutant chromosome of the pregnant female, k being an integer equal to or greater than one;   calculating, by the computer system, a second cutoff value for determining whether the fetus has inherited the normal allele at the locus, wherein the second cutoff value is derived at least from a second proportion of [k(1−Pf)]/[1+k−kPf)]; and   comparing, by the computer system, the parameter to at least one of the first and second cutoff values to determine a classification of whether the fetus has inherited the mutant allele or the normal allele.   
     
     
         2 . The method of  claim 1 , wherein the parameter is compared to the first and second cutoff values. 
     
     
         3 . The method of  claim 2  wherein the classifications include disease state, non-disease state, and non-classifiable. 
     
     
         4 . The method of  claim 1 , wherein obtaining the percentage Pf includes:
 correcting an experimentally derived percentage of fetal nucleic acid molecules in the biological sample with an expected statistical distribution of molecules in the plurality of reactions.   
     
     
         5 . The method of  claim 1 , wherein obtaining the percentage Pf includes:
 detecting a first allele in the reactions, wherein the first allele is shared by the pregnant female and fetus at a locus where the pregnant female is homozygous and the fetus is either heterozygous or hemizygous;   calculating a Poisson-corrected concentration Px with the equation [−ln((N−P1)/N)]*N, where N is the total number of reactions analyzed, P1 is the number of reactions positive for the first allele, and ln is the natural logarithm;   detecting a second allele in the reactions, wherein the second allele is specific to the fetus; and   calculating a Poisson-corrected concentration Py with the equation [−ln((N−P2)/N)]*N, where N is the total number of reactions analyzed, and P2 is the number of reactions positive for the second allele.   
     
     
         6 . The method of  claim 5 , wherein the second allele is on chromosome Y. 
     
     
         7 . The method of  claim 5 , wherein the first allele is on chromosome X. 
     
     
         8 . The method of  claim 5 , wherein the fetal-specific allele is a paternally-inherited allele on an autosome. 
     
     
         9 . The method of  claim 5 , wherein the fetal-specific allele includes a methylation marker specific to the fetus. 
     
     
         10 . The method of  claim 5 , further comprising:
 calculating Pf as [(2Py)/(Px+Py)]*100%.   
     
     
         11 . The method of  claim 1 , wherein the first and second cutoff values are determined using a sequential probability ratio test (SPRT) to determine whether the fetus has inherited the mutant or the normal nucleic acid sequence. 
     
     
         12 . The method of  claim 1 , wherein an allele at a polymorphic site linked to the mutant allele is located on the same maternal haplotype as the mutant allele, and wherein the probability of recombination between the polymorphic site and the mutant allele is less than 1%. 
     
     
         13 . The method of  claim 1 , wherein an allele at a polymorphic site linked to the normal allele is located on the same maternal haplotype as the normal allele, and wherein the probability of recombination between the polymorphic site and the mutant allele is less than 1%. 
     
     
         14 . The method of  claim 1 , wherein the reactions include any one or more of the following: sequencing reactions, optical analysis, and hybridization using a fluorescent probe, or nanopore sequencing. 
     
     
         15 . The method of  claim 1 , wherein a reaction is an amplification reaction. 
     
     
         16 . The method of  claim 15 , wherein the reactions include polymerase chain reactions. 
     
     
         17 . The method of  claim 15 , wherein an average concentration is less than one template molecule per reaction, and wherein a Poisson distribution is used in determining the percentage Pf of fetal nucleic acid molecules in the biological sample. 
     
     
         18 . The method of  claim 1 , wherein the biological sample is plasma, serum, or whole blood from a pregnant woman. 
     
     
         19 . The method of  claim 1 , further comprising:
 displaying, by the computer system, the classification of whether the fetus has inherited the mutant allele or the normal allele based on comparing the parameter to at least one of the first and second cutoff values.   
     
     
         20 . The method of  claim 1 , wherein the plurality of reactions is at least 1,000 reactions. 
     
     
         21 . The method of  claim 1 , further comprising:
 determining the fetus has inherited the mutant allele.   
     
     
         22 . The method of  claim 1 , wherein the X-linked mutation is a mutation related to hemophilia, Duchenne muscular dystrophy, X-linked adrenoleukodystrophy, Becker muscular dystrophy, choroideremia, Hunter syndrome, Lesch Nyhan syndrome, Norrie's syndrome, or ornithine transcarbamylase deficiency. 
     
     
         23 . The method of  claim 1 , wherein the X-linked mutation is a mutation related to hemophilia, and
 the method further comprising:
 determining the fetus has inherited the mutant allele. 
   
     
     
         24 . The method of  claim 1 , wherein the first amount is less than 1160, and the method further comprises determining the fetus has inherited the mutant allele. 
     
     
         25 . The method of  claim 1 , wherein:
 the parameter is a first parameter;   the method further comprising:
 determining based on the first cutoff value and the second cutoff value that the fetus cannot be classified as inheriting the mutant allele and cannot be classified as inheriting the normal allele; 
 receiving, by the computer system, data from a second plurality of reactions, each reaction involving one or more nucleic acid molecules from the biological sample, wherein data from each of the one or more second pluralities of reactions includes:
 a third set of quantitative data indicating a third amount of the mutant allele at the locus; and 
 a fourth set of quantitative data indicating a fourth amount of the normal allele at the locus; 
 
 determining, by the computer system, a second parameter from the first amount, the second amount, the third amount, and the fourth amount, wherein the second parameter represents a relative amount between the sum of the first amount and the third amount and the sum of the second amount and the fourth amount; 
 comparing, by the computer system, the second parameter to at least one of the first and second cutoff values to classify the fetus as inheriting either the mutant allele or the normal allele. 
   
     
     
         26 . The method of  claim 25 , wherein the first plurality of reactions and the second plurality of reactions total to less than or equal to 13,770 reactions. 
     
     
         27 . The method of  claim 25 , wherein:
 the first cutoff value is based on a total number of reactions, and the second cutoff value is based on the total number of reactions,   the method further comprising:
 updating the first cutoff value based on a total number of the first plurality of reactions and the second plurality of reactions, and 
 updating the second cutoff value based on the total number of the first plurality of reactions and the second plurality of reactions. 
   
     
     
         28 . The method of  claim 26 , wherein:
 updating the first cutoff value comprises using sequential probability ratio test (SPRT), and   updating the second cutoff value comprises using SPRT.   
     
     
         29 . The method of  claim 1 , wherein:
 the classification comprises a probability of accuracy,   the probability of accuracy determined by how much the parameter exceeds at least one of the first and second cutoff values.   
     
     
         30 . The method of  claim 1 , further comprising:
 causing the plurality of reactions to be performed.   
     
     
         31 . A computer program product comprising a non-transitory computer readable medium storing a plurality of instructions for controlling an apparatus to perform the method of  claim 1 . 
     
     
         32 . A method for determining whether a male fetus of a pregnant female has an X-linked mutation, the method comprising:
 receiving, by a computer system, data from a plurality of reactions, each involving one or more nucleic acid molecules from a biological sample, the biological sample including cell-free nucleic acid molecules from the pregnant female and from the male fetus,
 wherein the pregnant female is homozygous for an allele at a locus on the X chromosome, has a mutation of an amplification of the allele on a mutant X chromosome, the mutant X chromosome having a normal copy of the allele at the locus and one or more additional copies of the allele, and has a normal X chromosome having a normal copy of the allele at the locus, 
   wherein the data includes:
 a first set of quantitative data indicating a first amount of an additional junction created by the one or more additional copies of the allele; and 
 a second set of quantitative data indicating a second amount of a normal junction created by the normal copy of the allele on both X chromosomes; 
   determining, by the computer system, a parameter from the first amount and the second amount, wherein the parameter represents a relative amount between the first and second amounts;   obtaining, by the computer system, a percentage Pf of fetal nucleic acid molecules in the biological sample;   calculating, by the computer system, a first cutoff value for determining whether the fetus has inherited the mutant X chromosome, wherein the first cutoff value is derived at least from a first proportion of n/(n+1−Pf), where n is the number of additional copies of the allele, n being an integer equal to or greater than one;   calculating, by the computer system, a second cutoff value for determining whether the fetus has inherited the normal X chromosome, wherein the second cutoff value is derived at least from a second proportion of [n(1−Pf)/[n+2−Pf(n+1)]; and   comparing, by the computer system, the parameter to at least one of the first and second cutoff values to determine a classification of whether the fetus has inherited the mutant X chromosome or the normal X chromosome.   
     
     
         33 . The method of  claim 32 , further comprising:
 determining the fetus has inherited the mutant X chromosome.   
     
     
         34 . The method of  claim 33 , wherein the mutant X chromosome is related to hemophilia. 
     
     
         35 . The method of  claim 32 , further comprising:
 causing the plurality of reactions to be performed.   
     
     
         36 . A method for determining whether a male fetus of a pregnant female has an X-linked mutation, wherein the pregnant female is heterozygous for a mutation and a normal allele at a target region on the X chromosome, wherein the mutation is a deletion or an amplification of the target region, the method comprising:
 receiving, by a computer system, data from a plurality of reactions, each involving one or more nucleic acid molecules from a biological sample, the biological sample including cell-free nucleic acid molecules from the pregnant female and from the male fetus, wherein the data includes:
 a first set of quantitative data indicating a first amount of the nucleic acid molecules that are from the target region; and 
 a second set of quantitative data indicating a second amount of the nucleic acid molecules that are from a reference region on the X chromosome; 
   determining, by the computer system, a parameter from the first amount and the second amount, wherein the parameter represents a relative amount between the first and second amounts;   obtaining, by the computer system, a percentage Pf of fetal nucleic acid molecules in the biological sample;   calculating, by the computer system, a first cutoff value for determining whether the fetus has inherited the mutation, the first cutoff value being dependent on the percentage Pf;   calculating, by the computer system, a second cutoff value for determining whether the fetus has inherited the normal allele, the second cutoff value being dependent on the percentage Pf; and   comparing, by the computer system, the parameter to at least one of the first and second cutoff values to determine a classification of whether the fetus has inherited the mutation or the normal allele.   
     
     
         37 . The method of  claim 36 , wherein the mutation is an amplification, wherein the first cutoff value is determined based on the assumption that a ratio of the first amount to the second amount is increased when compared with a corresponding ratio of a non-pregnant woman carrying the same amplification mutation, and the second cutoff value is based on the assumption that the ratio of the first amount to second amount is decreased when compared with the corresponding ratio of a non-pregnant woman carrying the same amplification mutation. 
     
     
         38 . The method of  claim 36 , wherein the mutation is a deletion, wherein the second cutoff value is determined based on the assumption that a ratio of the first amount to the second amount is increased when compared with a corresponding ratio of a non-pregnant woman carrying the same deletion mutation, and the first cutoff value is based on the assumption that the ratio of the first amount to the second amount is decreased when compared with the corresponding ratio of a non-pregnant woman carrying the same deletion mutation. 
     
     
         39 . The method of  claim 36 , wherein the mutation is a deletion, wherein the second cutoff value is derived at least from a first proportion of 1/(2−Pf), and wherein the first cutoff value is derived at least from a second proportion of (1−Pf)/(2−Pf). 
     
     
         40 . The method of  claim 36 , wherein the mutation is a duplication, wherein the second cutoff value is derived at least from a first proportion of (3−Pf)/(2−Pf), and wherein the first cutoff value is derived at least from a second proportion of (3−2Pf)/(2−Pf). 
     
     
         41 . The method of  claim 36 , wherein obtaining the percentage Pf includes:
 correcting an experimentally derived percentage of fetal nucleic acid molecules in the biological sample with an expected statistical distribution of molecules in the plurality of reactions.   
     
     
         42 . The method of  claim 36 , further comprising:
 determining the fetus has inherited the mutation.   
     
     
         43 . The method of  claim 42 , wherein the mutation is related to hemophilia. 
     
     
         44 . The method of  claim 36 , further comprising:
 causing the plurality of reactions to be performed.   
     
     
         45 . A method of obtaining a percentage Pf of fetal nucleic acid molecules in a biological sample from a female pregnant with a fetus, the method comprising:
 receiving, by a computer system, data from a plurality of reactions, each involving a plurality of nucleic acid molecules from the biological sample, the biological sample including cell-free nucleic acid molecules from the pregnant female and from the fetus;   detecting, by the computer system, a first allele in the reactions, wherein the first allele is shared by the pregnant female and fetus at a locus where the pregnant female is homozygous and the fetus is either heterozygous or hemizygous, wherein detecting the first allele comprises aligning sequence tags to a reference genome to identify tags aligning to the first allele;   calculating, by the computer system, a corrected concentration Px of the first allele based on a number of reactions positive for the first allele, where Px is corrected for an expected statistical distribution of the first allele in the plurality of reactions;   detecting, by the computer system, a second allele in the reactions, wherein the second allele is specific to the fetus, wherein detecting the second allele comprises aligning sequence tags to a reference genome to identify tags aligning to the second allele;   calculating, by the computer system, a corrected concentration Py of the second allele based on a number of reactions positive for the second allele, where Py is corrected for an expected statistical distribution of the second allele in the plurality of reactions; and   calculating, by the computer system, Pf using [(2Py)/(Px+Py)].   
     
     
         46 . The method of  claim 45 , wherein Pf equals [(2Py)/(Px+Py)]*100%. 
     
     
         47 . The method of  claim 45 , wherein the statistical distribution is Poisson, and wherein the Poisson-corrected concentration Px uses the equation [−ln((N−P1)/N)]*N, where N is the total number of reactions analyzed, P1 is the number of reactions positive for the first allele, and ln is the natural logarithm, and wherein the Poisson-corrected concentration Py uses the equation [−ln((N−P2)/N)]*N, where N is the total number of reactions analyzed, and P2 is the number of reactions positive for the second allele. 
     
     
         48 . The method of  claim 45 , wherein the data includes:
 a first set of quantitative data indicating a first amount of an allele at a polymorphic site linked to the first allele; and   a second set of quantitative data indicating a second amount of an allele at a polymorphic site linked to the second allele, the method further comprising:   determining a parameter from the two data sets;   determining a first cutoff value for determining whether the fetus has inherited a mutant nucleic acid sequence, wherein the first cutoff value is determined based on the percentage Pf;   determining a second cutoff value for determining whether the fetus has inherited the normal nucleic acid sequence, wherein the second cutoff value is determined based on the percentage Pf;   comparing the parameter to at least one of the first and second cutoff values; and   based on the comparison, determining a classification of whether the fetus has inherited the mutant or the normal nucleic acid sequence.   
     
     
         49 . The method of  claim 45 , further comprising:
 causing the plurality of reactions to be performed.