High resolution non-invasive detection of a fetal microdeletion using the GCREM algorithm

Abstract

Background/objective: The non-invasive prenatal detection of fetal microdeletions becomes increasingly challenging as the size of the mutation decreases, with current practical lower limits in the range of a few megabases. Our goals were to explore the lower limits of microdeletion size detection via non-invasive prenatal tests using Minimally Invasive Karyotyping (MINK) and introduce/evaluate a novel statistical approach we recently developed called the GC Content Random Effect Model (GCREM).

Methods: Maternal plasma was obtained from a pregnancy affected by a 4.2-Mb fetal microdeletion and three normal controls. Plasma DNA was subjected to capture an 8-Mb sequence spanning the breakpoint region and sequence. Data were analyzed with our published method, MINK, and a new method called GCREM.

Results: The 8-Mb capture segment was divided into either 38 or 76 non-overlapping regions of 200 and 100 Kb, respectively. At 200 Kb resolution, using GCREM (but not MINK), we obtained significant adjusted p-values for all 20 regions overlapping the deleted sequence, and non-significant p-values for all 18 reference regions. At 100 Kb resolution, GCREM identified significant adjusted p-values for all but one 100-Kb region located inside the deleted region.

Conclusion: Targeted sequencing and GCREM analysis may enable cost effective detection of fetal microdeletions and microduplications at high resolution.

Figure 1. Difference between the fitted and observed normalized log tag count in PL565 for reads aligned to the 36 regions of chr12 using the GCREM algorithm with 3 reference libraries

Each region is 200kb long. The regions between the two vertical dashed lines indicate the position where the microdeletion occurs. The two horizontal dashed lines represent the standard deviation of the difference. For each region with complete or partial deletion (chr12:24380000~28760000), the GCREM test is using the 16 regions covering chr12:22620000~24360000 and chr12:28780000~30520000 as the control regions. For each of the 16 regions with no deletion, the test is using the other 15 regions with no deletion as control regions. The three plots, from top to bottom, are based on the full, one-half, and one-quarter of the PL565 sequence reads respectively.

Figure 2. Adjusted p values (using Holm’s method) of the GCREM tests for PL565 on the 36 regions of chr12

Each region is 200kb long. The regions between the two vertical dashed lines are where the microdeletion occurs. For each region with complete or partial deletion (chr12:24380000~28760000), the GCREM test is using the 16 regions covering chr12:22620000~2436000 and chr12:28780000~30520000 as the control regions. For each of the 16 regions with no deletion, the test is using the other 15 regions with no deletion as control regions. The three plots, from top to bottom, are based on the full, one-half, and one-quarter of the PL565 sequence reads respectively.

Figure 3A. Medians of adjusted p values (using Holm’s method) of the MINK tests for PL565 on the 36 regions of chr12

The regions between the two vertical dashed lines are where the microdeletion occurs. Each region is 200kb long. For each region with complete or partial deletion (chr12:24380000~28760000), three MINK tests, each against a different reference library, were performed, using the 16 regions covering chr12:22620000~2436000 and chr12:28780000~30520000 as the control regions. For each of the 16 regions with no deletion, three MINK tests are using the other 15 regions with no deletion as control regions. For each region, the median of the adjusted p values reported by the three MINK tests is plotted. The three plots, from top to bottom, are based on the full, one-half, and one-quarter of the PL565 sequence reads respectively.

Figure 3B. Medians of unadjusted p values of the MINK tests for PL565 on the 36 regions of chr12

The regions between the two vertical dashed lines are where the microdeletion occurs. The three plots, from top to bottom, are based on the full, one-half, and one-quarter of the PL565 sequence reads respectively.

Figure 4. Adjusted p values (using Holm’s method) of the GCREM tests for PL565 on the 74 regions of chr12

Each region is 100kb long. The regions between the two vertical dashed lines are where the microdeletion occurs. For each region with complete or partial deletion, the GCREM test is using the 35 regions with no deletion, covering chr12:22510000~24370000 and chr12:28670000~30640000, as the control regions. For each of the 35 regions with no deletion, the test is using the other 34 regions with no deletion as control regions. The three plots, from top to bottom, are based on the full, one-half, and one-quarter of the PL565 sequence reads respectively.