Exonic duplication CNV of NDRG1 associated with autosomal-recessive HMSN-Lom/CMT4D

Abstract

Purpose: Copy-number variations as a mutational mechanism contribute significantly to human disease. Approximately one-half of the patients with Charcot-Marie-Tooth (CMT) disease have a 1.4 Mb duplication copy-number variation as the cause of their neuropathy. However, non-CMT1A neuropathy patients rarely have causative copy-number variations, and to date, autosomal-recessive disease has not been associated with copy-number variation as a mutational mechanism.

Methods: We performed Agilent 8 × 60 K array comparative genomic hybridization on DNA from 12 recessive Turkish families with CMT disease. Additional molecular studies were conducted to detect breakpoint junctions and to evaluate gene expression levels in a family in which we detected an intragenic duplication copy-number variation.

Results: We detected an ~6.25 kb homozygous intragenic duplication in NDRG1, a gene known to be causative for recessive HMSNL/CMT4D, in three individuals from a Turkish family with CMT neuropathy. Further studies showed that this intragenic copy-number variation resulted in a homozygous duplication of exons 6-8 that caused decreased mRNA expression of NDRG1.

Conclusion: Exon-focused high-resolution array comparative genomic hybridization enables the detection of copy-number variation carrier states in recessive genes, particularly small copy-number variations encompassing or disrupting single genes. In families for whom a molecular diagnosis has not been elucidated by conventional clinical assays, an assessment for copy-number variations in known CMT genes might be considered.

Figure 1. Array comparative genomic hybridization (aCGH) results of the NDRG1 gene region in family HOU1463 and breakpoint junction analysis

(a) aCGH segregation study within the family. Left column shows the pedigree with four affected (three black boxes indicate CMT4D, and the checkered box indicates the different, more severe neuropathy, and the intellectual disability of the phenotypically distinct sister) and three unaffected individuals born from apparently healthy individuals. Right column shows the figure of custom microarray (Agilent 8 × 60K) for all individuals available for study (II-1 was unavailable for molecular studies). Wt indicates wild type, Hmz indicates homozygous, and Htz indicates heterozygous alleles. (b) Graphic view of 16 exons (vertical black and red bars) of NDRG1. Red bars indicate the duplicated exons (exon 6 through 8). Size and orientation of NDRG1 is shown above the exons. (c) Breakpoint sequence analysis of the duplication. The proximal and distal sequences refer to reference sequence and to their position from the centromere. Proximal reference sequence and patient breakpoint sequences that match with proximal reference sequence are shown in red and distal reference sequence and patient breakpoint sequences that match with distal reference sequence are shown in green. Boxed sequences (purple) correspond to regions of microhomology and emphasize the sequence structure at breakpoint junction.

Figure 2. Polymerase chain reaction (PCR) results for copy-number variation (CNV) gain and breakpoint junction

(a) Pedigree of family HOU1463. Each individual is coded with a BAB number (white in respective gel lanes). The array results for each individual are represented by two black chromosome schematics, in which a red region on the q arm designates the duplication predicted by array, and a black chromosome represents no rearrangement. The presumed array genotype is under the chromosome schematics, as well. Wt indicates wild type, Hmz indicates Homozygous, and Htz indicates heterozygous alleles. (b) Long-range PCR for duplicated region by primer pair 3724-F1 and 3724-R1. Individuals with a homozygous duplication allele (BAB3724, BAB4148, and BAB4149) show a single band migrating at ~14.8 kb in size, individuals with heterozygous duplication (BAB4146, BAB4147, and BAB4151) have two bands at 14.8 and 7.4 kb, and individuals who only carry the wild-type allele (BAB4150 and BAB4152) have one 7.4 kb band. (c) Long-range PCR assay results for the breakpoint junction by primer pair 3724-2F1 and 3724-2R1. Individuals who carry at least one duplicated segment have a 1 kb amplicon and individuals who are wild type do not, as expected, show any breakpoint junction fragment. (d) Array comparative genomic hybridization image showing location and orientation of primers to detect breakpoint junction and to amplify the entire duplicated segment. F1 and R1 primers amplify the entire product. 2F1 and 2R1 primers are placed inside the duplication in outward-facing orientation to obtain the breakpoint junction.

Figure 3. Sequence analysis for cDNA and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) study for NDRG1

(a) Sanger sequence results of cDNA from the control and affected individuals. In healthy individuals, exon 8 is followed by exon 9 and in affected individuals exon 8 is followed by exon 6. (b) Analysis of the resultant sequence at the amino acid level. The duplication causes a frame shift mutation, altering the amino acid sequence for several residues before a premature termination codon. (c) Expression levels in blood were measured by quantitative RT-PCR using the TaqMan gene expression assay in triplicate and normalized to TBP. We observed a decrease in expression, 0.12- to 0.18-fold in patient samples (BAB3724 and BAB4149) and 0.6- to 0.61-fold in carriers (BAB4146 and BAB4147), compared with normal expression controls. We found an obviously decreased expression in both patients and carriers compared with control healthy individuals. *P < 0.01.

Figure 4. NDRG1 resides in an ~22 Mb region of absence of heterozygosity (AOH) shared by all affected individuals

The figure depicts single-nucleotide polymorphism array data for the entirety of chromosome 8. Illumina Genome Studio software predicted AOH regions >0.25 Mb (shaded in gray). All affected individuals in the pedigree share a minimum overlapping region of ~21.9 Mb of AOH that encompasses the NDRG1 gene locus. All unaffected family members lacked a predicted AOH region that overlapped this gene. The location of NDRG1 is depicted at the bottom of the figure, and its location for each individual in the pedigree is denoted with the red vertical line.