PMC4804755_01
A) Pedigree of Family NG1278: Pedigree of family NG1278 demonstrating the affected siblings. Note the consanguinity of the parents.. B) Sural Nerve Biopsy of Patient NG1278-1: 40x magnification image of a representative section of sural nerve tissue obtained via biopsy from patient NG1278-1. There is general axonal loss and decreased myelination; # indicates the marked decrease in small myelinated fibers; * indicates the segmentally decreased number of large diameter myelinated fibers; non-myelinated fibers also demonstrate a slight decrease in numbers, but appear relatively spared (arrow).. C) Brain MRI of Patient NG1278-1: Sagittal T1 weighted brain MRI demonstrating a thin spinal cord. No cortical abnormalities were noted.. D) Sequence Alignment: Whole-exome sequencing results for patient NG1278-1 demonstrating the homozygous 4 base pair deletion in NGLY1 - delTTGA. The top line represents the wild-type reference sequence. The subsequent lines below the reference lines depict the results from exome sequencing. Each line represents a distinct coverage read. Coverage achieved for this region was 15x.. E) Sanger Sequencing Results: Chromatographs obtained via Sanger sequencing analysis of the two patients and their parents. Sanger sequencing of wild-type control DNA was also performed. Note that the mutation identified via whole-exome sequencing was confirmed as being homozygous versus heterozygous, in the two affected siblings versus their parents, respectively. The bases outlined in red in the wild-type sequence indicate the mutated base pairs.. F) Schematic Representation of N-glycanase 1: Figure shows the functional and conserved domains. Arrow head indicates location of the identified mutation.. G) CNV Analysis of NG1278-1: The log ratio comparing NG1278-1 and control sequence depths of coverage for each exon are depicted as gray dots. The black lines demonstrate regions of segmented copy neutral events, green lines are segmented deletion events and red lines are amplification events.
nihms627109f1.jpg
PMC4804755_02_nihms627109f1.jpg
F1
However, this variant did not segregate with the disease phenotype and was found to be heterozygous in his affected sibling (NG1278-2) (Supplementary Figure 1). This mutation segregated in the expected pattern in the family with the affected sister being homozygous and both unaffected parents being heterozygous (Figure 1E). This NGLY1 mutation (Figure 1F) has neither been previously reported in the dbSNP, NHLBI GO ESP Exome Variant Server, or 1000 Genomes databases, nor has it been observed within a cohort of 3,000 subjects with non-neurological diseases who were whole-exome sequenced at Yale School of Medicine. In addition, copy number variation (CNV) analysis, based on exome sequencing of the index case, demonstrated no disease causing large-scale amplifications, deletions, or loss of heterozygosity (aside from aforementioned inherited region of homozygosity) within the coding regions of the entire genome (Figure 1G).
PMC4804755_02
A) Pedigree of Family NG1278: Pedigree of family NG1278 demonstrating the affected siblings. Note the consanguinity of the parents.. B) Sural Nerve Biopsy of Patient NG1278-1: 40x magnification image of a representative section of sural nerve tissue obtained via biopsy from patient NG1278-1. There is general axonal loss and decreased myelination; # indicates the marked decrease in small myelinated fibers; * indicates the segmentally decreased number of large diameter myelinated fibers; non-myelinated fibers also demonstrate a slight decrease in numbers, but appear relatively spared (arrow).. C) Brain MRI of Patient NG1278-1: Sagittal T1 weighted brain MRI demonstrating a thin spinal cord. No cortical abnormalities were noted.. D) Sequence Alignment: Whole-exome sequencing results for patient NG1278-1 demonstrating the homozygous 4 base pair deletion in NGLY1 - delTTGA. The top line represents the wild-type reference sequence. The subsequent lines below the reference lines depict the results from exome sequencing. Each line represents a distinct coverage read. Coverage achieved for this region was 15x.. E) Sanger Sequencing Results: Chromatographs obtained via Sanger sequencing analysis of the two patients and their parents. Sanger sequencing of wild-type control DNA was also performed. Note that the mutation identified via whole-exome sequencing was confirmed as being homozygous versus heterozygous, in the two affected siblings versus their parents, respectively. The bases outlined in red in the wild-type sequence indicate the mutated base pairs.. F) Schematic Representation of N-glycanase 1: Figure shows the functional and conserved domains. Arrow head indicates location of the identified mutation.. G) CNV Analysis of NG1278-1: The log ratio comparing NG1278-1 and control sequence depths of coverage for each exon are depicted as gray dots. The black lines demonstrate regions of segmented copy neutral events, green lines are segmented deletion events and red lines are amplification events.
nihms627109f1.jpg
PMC4804755_02_nihms627109f1.jpg
F1
However, this variant did not segregate with the disease phenotype and was found to be heterozygous in his affected sibling (NG1278-2) (Supplementary Figure 1). This mutation segregated in the expected pattern in the family with the affected sister being homozygous and both unaffected parents being heterozygous (Figure 1E). This NGLY1 mutation (Figure 1F) has neither been previously reported in the dbSNP, NHLBI GO ESP Exome Variant Server, or 1000 Genomes databases, nor has it been observed within a cohort of 3,000 subjects with non-neurological diseases who were whole-exome sequenced at Yale School of Medicine. In addition, copy number variation (CNV) analysis, based on exome sequencing of the index case, demonstrated no disease causing large-scale amplifications, deletions, or loss of heterozygosity (aside from aforementioned inherited region of homozygosity) within the coding regions of the entire genome (Figure 1G).