Bank Research

A novel missense NMNAT1 mutation identified in a consanguineous family with Leber congenital amaurosis by targeted next generation sequencing

Leber congenital amaurosis (LCA; MIM 204000) is a group of inherited retinal dystrophies that is the earliest occurring and most se- vere form of visual impairment within the first year of life. Its clinical features include loss of vision from birth, sensory nystagmus, amaurotic pupils and absent electrical signals on an electroretinogram (ERG). Being a rare form of childhood blindness, the estimated prevalence of LCA is between 1/81,000 and 1/30,000 (Stone, 2007; Koenekoop, 2004). LCA is predominantly inherited in an autosomal recessive manner, but some autosomal dominant families have been reported


(Tzekov et al., 2001; Nichols et al., 2010). To date, mutations in 21 genes have been identified to be responsible for LCA, which only ex- plains the cause of a part of LCA cases (Retinal Information Network (RetNet, the remainder has been studied in order to identify new causative genes (den Hollander et al., 2008).

Elucidating the genetic basis of LCA is necessary for guiding the potential therapies in the future, just as the clinical gene therapies for LCA caused by RPE mutations (Maguire et al., 2009; Jacobson et al., 2012). Direct mutational screening of all known genes involved in retinal diseases requires excessive labor and time as well as high cost. Because o fscreening multiple gene simultaneously, targeted next generation sequencing has become a rapid and effective method in performing an accurate molecular diagnosis for genetically heterogeneous diseases, such as LCA.

In this study, targeted next generation sequencing was performed in two affected siblings from a consanguineous Chinese family with suspected LCA. The purpose of our study was to detect the causative variants in this family and the information we had from this study can be used for mutation screening in the future.


  1. Materials and methods

2.1. Ethics statement and study subjects

The study was approved by Ethic Committee of (BGI)-Shenzhen (No. 13066) and written informed consent was obtained from all participants before the study. All procedures were carried out according to the tenets of the Declaration of Helsinki. The subjects of this study included a consanguineous Han Chinese family with suspected LCA from Hunan province in China (Fig. 1), and 300 healthy controls. The proband (II:2), a 24-year-old male patient, is the second child of the healthy parents who are first cousins. The proband had severe, early-onset vision loss since birth (Table 1). The macular coloboma was found in fundus photograph of the proband (Fig. 2). The proband’s younger brother (II:5), a 10-year-old boy, had the same clinical symptoms after birth. The controls denied any personal and family history of known inherited diseases.

2.2. Targeted region capture and next generation sequencing analysis

2.2.1. Targeted region capture and next generation sequencing

Venous blood samples were obtained from the two siblings and their parents. Genomic DNA was isolated from leukocytes of blood samples with the QIAamp Blood DNA mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Targeted next generation sequencing was performed on DNA from the two siblings (II:2 and II:5) by BGI-Shenzhen, China, with a capture panel which was designed to capture the coded exons of 283 genes associated with hereditary eye diseases including 19 known LCA disease-causing genes (AIPL1, CABP4, CEP290, CRB1, CRX, GUCY2D, IMPDH1, IQCB1, LCA5, LRAT, OTX2, RD3, RDH12, RPE65, RPGRIP1, SPATA7, TULP1, KCNJ13, NMNAT1). The genomic

DNA was randomly fragmented into 200–300 bp by sonication (Covaris S2, Massachusetts, USA) and added an “A” residue, then amplified by ligation-mediated PCR (LM-PCR). The products were purified and hybridized to the custom NimbleGen EZ choice probe library. After washing non-hybridized fragments and eluting target DNA fragment, each captured library was loaded on Hiseq2000 platform for highthroughput sequencing. Raw image files were processed by Illumina Pipeline v1.7 for base calling with default parameters and the sequences of each individual were generated as 90 bp paired-end reads. The sequences for this array are available in the Consensus Coding Sequence Region (CCDS) database. Buy NMN

2.2.2. Read mapping and variant analysis

The human reference genome and its gene annotation, were downloaded from the UCSC database (, version hg19 (NCBI build 37.1). After removing the duplicated reads, the sequence reads were mapped back to the reference sequences using BWA (Burrows Wheeler Aligner) Multi-Vision software package. The software SOAPsnp v 1.05 was used to assemble the consensus sequence and data were shown as a list of single nucleotide variants


(SNVs) (Li and Durbin, 2009), while insertions and deletions (Indels) were identified using the Samtools (tools for alignments in the SAM format) Version: 0.1.18 ( All changes were filtered against several databases including dbSNP, 1000 genome project, HapMap database, ExAc and ESP6500. Sanger sequencing was then used to determine whether the identified disease-causing variants co-segregated with the phenotype in the family.


Published by admin, on July 28th, 2018 at 1:55 pm. Filled under: Uncategorized. | No Comments |