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. 2019 Jul 3;105(1):166-176.
doi: 10.1016/j.ajhg.2019.05.013. Epub 2019 Jun 6.

Expansion of Human-Specific GGC Repeat in Neuronal Intranuclear Inclusion Disease-Related Disorders

Yun Tian  1 , Jun-Ling Wang  2 , Wen Huang  3 , Sheng Zeng  2 , Bin Jiao  2 , Zhen Liu  2 , Zhao Chen  2 , Yujing Li  4 , Ying Wang  5 , Hao-Xuan Min  6 , Xue-Jing Wang  7 , Yong You  8 , Ru-Xu Zhang  9 , Xiao-Yu Chen  10 , Fang Yi  11 , Ya-Fang Zhou  11 , Hong-Yu Long  2 , Chao-Jun Zhou  2 , Xuan Hou  2 , Jun-Pu Wang  5 , Bin Xie  5 , Fan Liang  6 , Zhuan-Yi Yang  10 , Qi-Ying Sun  11 , Emily G Allen  4 , Andrew Mark Shafik  4 , Ha Eun Kong  4 , Ji-Feng Guo  2 , Xin-Xiang Yan  2 , Zheng-Mao Hu  12 , Kun Xia  12 , Hong Jiang  13 , Hong-Wei Xu  11 , Ran-Hui Duan  12 , Peng Jin  14 , Bei-Sha Tang  15 , Lu Shen  16
Affiliations

Expansion of Human-Specific GGC Repeat in Neuronal Intranuclear Inclusion Disease-Related Disorders

Yun Tian et al. Am J Hum Genet. .

Abstract

Neuronal intranuclear inclusion disease (NIID) is a slowly progressing neurodegenerative disease characterized by eosinophilic intranuclear inclusions in the nervous system and multiple visceral organs. The clinical manifestation of NIID varies widely, and both familial and sporadic cases have been reported. Here we have performed genetic linkage analysis and mapped the disease locus to 1p13.3-q23.1; however, whole-exome sequencing revealed no potential disease-causing mutations. We then performed long-read genome sequencing and identified a large GGC repeat expansion within human-specific NOTCH2NLC. Expanded GGC repeats as the cause of NIID was further confirmed in an additional three NIID-affected families as well as five sporadic NIID-affected case subjects. Moreover, given the clinical heterogeneity of NIID, we examined the size of the GGC repeat among 456 families with a variety of neurological conditions with the known pathogenic genes excluded. Surprisingly, GGC repeat expansion was observed in two Alzheimer disease (AD)-affected families and three parkinsonism-affected families, implicating that the GGC repeat expansions in NOTCH2NLC could also contribute to the pathogenesis of both AD and PD. Therefore, we suggest defining a term NIID-related disorders (NIIDRD), which will include NIID and other related neurodegenerative diseases caused by the expanded GGC repeat within human-specific NOTCH2NLC.

Keywords: GGC repeat expansions; NOTCH2NLC; linkage analysis; long-read genome sequencing; neuronal intranuclear inclusion disease; whole-exome sequencing.

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Figures

Figure 1
Figure 1
Identification of Expanded GGC Repeat within NOTCH2NLC in Neuronal Intranuclear Inclusion Disease (A) Pedigrees of neuronal intranuclear inclusion disease (NIID)-affected families and corresponding individual genotypes. (B) Genetic linkage analysis indicated maximum logarithm of odds (LOD) scores 3.184 in chromosome 1, a 49.8-Mb region at 1p13.3-q23.1 (chr1:109260034-159016186). (C and D) GGC expansions detected by LRS. Nanopore electric signal (C) from subject F1-IV:15 indicated GGC expansions in the lower lane compared to the normal allele in the upper lane. More than ten reads covering the causative region were seen in the Integrative Genomics Viewer for subject F1-IV:15. Two reads were determined to carry the “insertion” variation (chr1:149390803-149390842, hg38 version), corresponding to the GGC triplet expansion in NOTCH2NLC (D). (E) Schematic representation of the causal variant in NOTCH2NLC: a certain number of GGC triplets exist in 5′ UTR of NOTCH2NLC in healthy individuals, and large expanded GGC triplets are present in affected individuals.
Figure 2
Figure 2
Validation of Expanded GGC Repeats and Variations of GGC Repeat Size among Normal Individuals (A–D) Representative electropherogram of the RP-PCR assay and the GC-PCR assay showed abnormal repeat expansion in affected individuals (C and D) and negative result in control subjects (A and B). (E) Size distribution of GGC repeat among healthy control subjects, which are usually less than 40 repeats.
Figure 3
Figure 3
Expanded GGC Repeats Are Associated with Dementia and Parkinsonism (A) Flow chart of GGC repeat expansion screening in a cohort of families with neurodegenerative disorders. (B) Pedigrees of AD-affected and parkinsonism-affected families that carry expanded GGC repeats in NOTCH2NLC.
Figure 4
Figure 4
Methylation and Expression at NOTCH2NLC Locus (A) Methylation status across expanded GGC repeats region was determined using LRS data from seven affected individuals (F1-IV:7, F1-IV:15, F2-II:3, F4-II:2, F5-II:1, F5-II:4, F9-II:6) and three healthy control subjects, and no significant methylation difference was detected between NIID-affected case subjects and control subjects. Wald test was performed for statistical analysis; ∗p < 0.05. (B) NOTCH2NLC expression level in both NIID-affected case subjects (NIID) and normal control subjects (Control). The ezDNase-treated total RNA isolated from the blood of both NIID-affected case subjects and control subjects was reversely transcribed into cDNA followed by quantitative PCR. GAPDH was used as internal control. Error bars represent the SD; Student’s t test was performed for statistical analysis; ∗p < 0.05. p = 0.776 (ns). (C) Expression levels of NOTCH2 and three NOTCH2NL paralogs (NOTCH2NLA, NOTCH2NLB, and NOTCH2NLC) in human adult cortex detected by RNA-seq. Significant differences were observed in the expression levels of these four genes. Shown are the normalized gene expression levels. Error bars represent the SD. (D) Dynamic change of NOTCH2NLC expression in human brain during aging. Relative expression levels of both NOTCH2NLB (left) and NOTCH2NLC (right) in DLPFC region of human brain during aging are shown. The total RNA isolated from the DLPFC of human postmortem brains from 4-, 15-, 36-, and 60-year-old subjects were used for quantitative RT-PCR with GAPDH as internal control. Error bars represent the SD.
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