Processes of identifying and characterizing X-linked disorders
The present invention relates to processes for characterizing and screening for the existence or predisposition to X-linked disorders associated with changes in X-chromosome inactivation. The present invention also relates to processes of reducing a disease phenotype associated with an X-linked disorder in a female subject.
1. A process of characterizing X chromosome inactivation (XCI) in a female subject, the process comprising:
(a) obtaining a first biological sample comprising nucleic acids from the female subject, a second biological sample comprising nucleic acids from the biological mother of the female subject, and a third biological sample comprising nucleic acids from the biological father of the female subject;
(b) isolating the nucleic acids from the first biological sample, the second biological sample, and the third biological sample;
(c) performing whole genome sequencing or whole exome sequencing by next generation sequencing of the isolated nucleic acids from the second biological sample, and the third biological sample;
(d) identifying from the whole genome sequencing or whole exome sequencing a plurality of X-linked variant alleles, wherein the female subject is heterozygous for the X-linked variant alleles;
(e) performing whole transcriptome sequencing by next generation sequencing of the isolated nucleic acids from the first biological sample;
(f) measuring from the whole transcriptome sequencing allelic ratios of the X-linked variant alleles at each heterozygous locus; and
(g) characterizing an XCI ratio in the female subject by determining a combined mean allelic ratio for the X-linked variant alleles from the allelic ratios at each heterozygous locus.
2. The process of claim 1 , further comprising performing whole genome sequencing or whole exome sequencing by next generation sequencing of the isolated nucleic acids from the first biological sample.
3. The process of claim 1 , wherein the whole transcriptome sequencing is performed simultaneously with step (c) and comprises whole mRNA sequencing.
4. The process of claim 1 , wherein the isolated nucleic acids in step (c) comprises genomic DNA and the isolated nucleic acids in step (e) comprise total RNA or mRNA.
5. A process of identifying an X-linked disorder in a female subject, the process comprising:
(a) characterizing X chromosome inactivation (XCI) in the female subject according to the process of claim 1 ; and
(b) identifying an X-linked disorder in the female subject if the combined mean allelic ratio for the X-linked variant alleles is substantially skewed, wherein the combined mean allelic ratio is substantially skewed if expression of the X-linked variant alleles from the biological mother of the female subject compared to expression of the X-linked variant alleles from the biological father of the female subject differs by at least a ratio of 65:35.
6. The process of claim 5 , wherein the expression of at least one of the X-linked variant alleles differs by at least 80:20.
7. The process of claim 5 , wherein the X-linked disorder is at least partially caused by a change in mode and magnitude of XCI.
8. The process of claim 7 , wherein the mode of XCI is determined by phasing of the chromosome-wide X-linked heterozygous SNP alleles of the female subject.
9. The process of claim 7 , wherein the magnitude of XCI is determined in silico by computer simulation of phased or unphased chromosome-wide X-linked heterozygous SNP alleles of the female subject or the magnitude of XCI is determined by allele-specific expression analysis of phased or unphased chromosome-wide X-linked heterozygous SNP alleles of the female subject that are transcribed into mRNA.
10. The process of claim 9 , wherein the XCI ratio is estimated from phased heterozygous SNPs by a beta parametric model and the XCI ratio is estimated from unphased heterozygous SNPs by a semi-parametric model.
11. The process of claim 7 , wherein a combination of the change in mode and magnitude of XCI and the presence or absence of at least one of the X-linked variant alleles is directly associated with a phenotype for the X-linked disorder.
12. The process of claim 11 , wherein the X-linked disorder is a neurobehavioral condition manifesting at least an emotional instability, attention deficit, or delays in development and learning.
13. The process of claim 5 , wherein the X-linked variant alleles comprise a chromosomal deletion, amplification, or both.
14. The process of claim 13 , wherein a chromosomal deletion is detected by plotting log 2 differences,
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across chromosomes, where a log 2 difference of −1 means a heterozygous deletion in one of the copies, a −2 means a homozygous deletion of both copies, a +1 means a heterozygous copy gain, and a +2 means a homozygous copy gain of the chromosomal region.
15. The process of claim 14 , further comprising characterizing by co-segregation analysis of parental genotypes obtained from whole exome sequencing.
16. The process of claim 1 , further comprising simulating RNA sequencing reads in silico from chromosome-wide heterozygous SNP alleles of the sample and calculating an allelic skewedness ratio by analysis of phased or unphased chromosome-wide heterozygous SNP variant transcripts from the simulated data.
17. The process of claim 16 , wherein the simulation of RNA sequencing reads in silico comprises:
(a) introducing nucleotide changes into a reference chromosome by selecting a number of chromosome wide SNPs from a sample;
(b) separating randomly the SNPs from the reference chromosome of interest into two groups, one group analogous to variant alleles on a maternal chromosome referred to as pseudo-maternal variant SNPs and the other group analogous to variant alleles on a paternal chromosome referred to as pseudo-paternal variant SNPs;
(c) introducing the pseudo-maternal and pseudo-paternal variant SNPs into two separate X chromosome files and reducing the two files to greater than 500 bp regions that correspond to known transcripts according to human genome annotation Homo sapiens to obtain two separate transcriptome files;
(d) generating paired reads mapping to the two separate transcriptome files; and
(e) sub-sampling the paired reads randomly in various ratios and merging the randomly distributed reads into a single file.
18. The process of claim 1 , wherein the combined mean allelic ratio is determined with X-linked variant alleles having a minimum of 20× sequencing coverage.
19. The process of claim 1 , wherein chromosome-wide allelic expression analysis provides an accurate estimate of overall expression of each X chromosome copy and serves as the basis for XCI ratio measurement.