A novel Nance-Horan syndrome mutation identified by next-generation sequencing in a Chinese family

INTRODUCTION

Nance-Horan syndrome (NHS; MIM 302350), a rare X-linked syndrome characterized by congenital bilateral cataracts and dental abnormity, was first reported in 1974 by two studies independently

. So far, this syndrome has been reported in different ethnic groups and reveal variable clinical features

. Male patients manifest severe bilateral congenital nuclear cataracts, including opacity in the fetal nucleus and posterior Y-suture; thus, surgical intervention should be performed at an early age

. Other ophthalmological abnormalities include microcornea, microphthalmia and nystagmus. Non-ophthalmic abnormalities include dental anomalies (supernumerary maxillary incisors, screwdrivershaped incisors, and diastema), mental retardation and lateral brachymetacarpalia. As an X-linked hereditary disorder,heterozygous females exhibit similar but milder features than affected males, including posterior Y-sutural cataracts, little or no loss of vision and occasional dental abnormalities

.

This family included two affected males. And all family members were further investigated for clinical features to provide precise information. Considering the unbalanced incidence between male and female members,we suggest the mode of inheritance was X-linked.

布朗預(yù)測(cè)，不用幾年，消費(fèi)者將再也不會(huì)擔(dān)心基因編輯的農(nóng)作物與傳統(tǒng)農(nóng)作物之間的區(qū)別。在現(xiàn)代農(nóng)業(yè)中，這是最具有革命性的幾次創(chuàng)新之一。

SUBJECTS AND METHODS

金融體系的多樣性對(duì)于整個(gè)經(jīng)濟(jì)的發(fā)展有著極其重要的作用，同時(shí)也有著非同凡響的意義。而且在信息技術(shù)層次不斷發(fā)展的背景下，對(duì)于互聯(lián)網(wǎng)金融來說更是有著十分重要的影響作用。通過各種金融信息技術(shù)將金融資源配置、風(fēng)險(xiǎn)管理都納入到技術(shù)層次，為金融基礎(chǔ)體系的構(gòu)建打下堅(jiān)實(shí)的基礎(chǔ)，同時(shí)對(duì)于電子銀行、互聯(lián)網(wǎng)金融市場(chǎng)的發(fā)展有著十分正面的作用。但是，由于其本身就具有高風(fēng)險(xiǎn)的特征，結(jié)合在一起必定會(huì)為互聯(lián)網(wǎng)金融帶來更大的財(cái)務(wù)風(fēng)險(xiǎn)，亟待構(gòu)建相應(yīng)的防范體系及控制機(jī)制，才能夠?yàn)榛ヂ?lián)網(wǎng)金融的穩(wěn)健打下堅(jiān)實(shí)的基礎(chǔ)。

A Chinese family(family LZ) with the initial symptom of poor central vision was recruited from the First Affiliated Hospital of Nanjing Medical University (Figure 1). Eight family members,including four affected patients and four healthy siblings,participated in our study. All included members from family LZ received ophthalmic examinations with their medical histories collected. Systemic examinations were conducted on the four patients. Another 150 unrelated healthy controls free of major ocular diseases were also recruited. Peripheral venous blood samples were collected from all participants from family LZ and 150 additionally unrelated healthy controls free of major ocular diseases using 5 mL tubes containing ethylenediamantetetraacetic acid (EDTA). Genomic DNA isolation was performed using a QIAmp DNA blood kit(Qiagen, Valencia, CA, USA) per the manufacturer’s protocols.DNA samples were stored at -20°C before used.

Our study, conformed to the tenets of the Declaration of Helsinki, was approved and prospectively reviewed by the Ethics Committee on Human Research ofNanjing Medical University. Written informed consents were signed by the participants or their legal guardians before enrollment.

The NHS gene, located on Xp22.13, is expressed during the development of embryonic tissues, especially in midbrain,lens, retina, craniofacial mesenchyme and tooth

. And it is conserved among human and other vertebrate species

.It comprises 10 exons which encompass about 650 kb of genomic DNA, and at least 4 different isoforms resulting from alternative splicing

. NHS-A and NHS-1A are the two major isoforms transcribed from exon1, encoding 1630 amino acids and 1651 amino acids respectively. NHS-B, encoding 1335 amino acids, is transcribed from exon 1b and translated from exon 4. NHS-C, encoding 1453 amino acids, is transcribed and translated from exon 1a. The exact biological function of NHS protein is unclear. To date, more than 40 mutations associated with NHS have been reported, originating from China, Australia, India, the United Kingdom, the United States of America and Turkey

. Most of the identified mutations are nonsense or indel, while others are frameshift mutations,genomic rearrangements and missense mutations

. The underlying consequence of these mutations can be classified into two categories. One is function damaging, the other is aberrant cellular distribution. In this study, the mutation in the NHS gene produced a truncated NHS protein. In most cases,the premature protein can initiate the nonsense-mediated mRNA decay pathway (NMD) which is able to degrade the shortened mRNA and protect cells from potential toxic effects resulting from dominant negative or gain-of-function effects

. Some mRNAs with PTCs, however, can avoid this translation coupled quality control system, resulting in truncated proteins. Weather this mutation is able to provide truncated protein requires further investigation. It has been reported that nonsense mutation can also lead to disrupted NH

-terminus and COOH-terminus of NHS protein resulting in aberrant intercellular location in the epithelium of lens and retinas. As NHS proteins are co-located with tight junction protein ZO-1, this loss of subcellular localization can cause disease

.Considering this mutation happened from 3916 to 3919 and NH

-terminus can act as a label for protein localization, the abnormal subcellular localization may not happen in this pedigree.

在菲律賓、馬來西亞等地區(qū)的熱帶雨林里，生活著一種長著白色“胡須”的豬——須野豬。這種被稱為“胡子豬”的豬科動(dòng)物，有著棕灰色的皮毛和顯眼的“大胡子”，體長多在100—165厘米之間，為中型偶蹄目動(dòng)物。

RESULTS

NHS gene locates to Xp22.31 to p22.13b between short tandem repeat markers DXS1195 and DXS999

. It is highly conserved among human and other vertebrates including rat,mouse, and zebrafish. NHS protein plays an important role in the development of ocular lens, tooth, midbrain, thus its mutations can lead to congenital cataract, dental anomalies and, in some cases, mental retardation

. Previous studies have discovered more than 40 mutations in the

gene including frameshift mutations, nonsense mutations, missense mutations,deletion mutations and genomic rearrangements

. Here,we report a novel a frameshift deletion in the

gene(c.3916_3919del) and characterize the clinical features of a Chinese pedigree with this syndrome.

NHS is an X-linked inheritance pattern involving bilateral congenital cataracts, dental anomalies and craniofacial dysmorphisms

. Mild mental retardation has been reported in about 20% of affected patients

. The proband (LZ-IV:2)and his brother (LZ-IV:1) exhibited congenital cataract with microcornea and strabismus which were similar to NHS syndrome. In order to find out the underlying causes, we performed targeted NGS, and found a novel mutation in the NHS gene. The pedigree family was recalled for further examination after this identification. The two affected males manifested characteristic ocular clinical features of NHS.Other two female carriers manifested milder signs (posterior Y-sutural cataracts). So, the results of targeted NGS as well as clinical features indicated the existence of NHS mutation in this pedigree.

二是適當(dāng)放開項(xiàng)目承擔(dān)單位在項(xiàng)目預(yù)算執(zhí)行調(diào)整方面的權(quán)限。政策調(diào)整前，項(xiàng)目單位只在勞務(wù)費(fèi)、專家咨詢費(fèi)和管理費(fèi)以外的其他8項(xiàng)單項(xiàng)支出科目中，當(dāng)預(yù)算支出科目不超出核定預(yù)算的10%、或雖然超出10%但金額不低于5萬元的預(yù)算執(zhí)行調(diào)整具有自主權(quán)。政策調(diào)整后，在項(xiàng)目總預(yù)算不變的前提下,項(xiàng)目承擔(dān)單位可以自主調(diào)整直接費(fèi)用中的材料費(fèi)等5項(xiàng)開支科目的預(yù)算執(zhí)行，并且沒有調(diào)整額度的限制。同時(shí)，《通知》還賦予項(xiàng)目承擔(dān)單位在設(shè)備費(fèi)等6項(xiàng)費(fèi)用上擁有調(diào)減用于課題其他方面支出的權(quán)限。

整本書閱讀不同于篇章閱讀，篇章閱讀以教材為主，但整本書閱讀還需要大量的圖書資源，以便于師生從中挑選、閱覽、研討，因此，學(xué)校還需要供給足夠多的優(yōu)質(zhì)書冊(cè)。如設(shè)立圖書館、閱覽室，豐富現(xiàn)有的圖書儲(chǔ)備。又因?yàn)檎緯喿x教學(xué)不只是停留在課外閱讀方面，還應(yīng)該根據(jù)教學(xué)的需要組織一定的教學(xué)活動(dòng)，因此，還要提供一定的場(chǎng)地、道具等設(shè)施，保障教學(xué)工作順利進(jìn)行。

Two heterozygous female carriers in this family (LZ-II:2 and LZ-III:5) presented fine, punctate opacities outlining the posterior Y-suture without visual acuity affected. LZ-III:2 manifested bilateral cortical opacities. No facial or dental abnormalities related to NHS were observed in these two carriers.

Targeted NGS approach was selectively conducted on patients LZ-IV:1 and LZ-IV:2. Coverage of the targeted region was 98.53% for patient LZ-IV:1 and 98.61% for patient LZ-IV:2. The mean depth of targeted region for the two patients reached 133.31- and 160.88-fold, respectively. A total of 3672 (3274 SNPs and 398 Indels) variants were initially revealed by targeted NGS approach for patient LZ-IV:1, and 3844 (3444 SNPs and 400 Indels) for patient LZ-IV:2. Only two variants retained after the filtration against the 5 SNP databases and were shared by the two tested patients, including a missense substitution in the

gene (c.7181C＞T) and a frameshift deletion in the

gene (c.3916_3919del). Since the pedigree of family LZ suggested the possibilities of both autosomal dominant and X-linked inheritance patterns, we therefore tried to determine the disease causative mutation for this family in both manners. In the autosomal dominant model,

c.7181C＞T failed the co-segregation analysis and was thus discarded, while in the X-linked way,

c.3916_3919del was shared among all affected patients and was further proved absent in 150 additional normal controls(Figure 3). This deletion was predicted to alter the reading frame and generate a premature termination codon (PTC) after a new reading frame of 8 amino acids (p.Ser1306Thrfs*9).

DISCUSSION

Our study discovered a Chinese family with NHS syndrome and identified a novel NHS frameshift mutation (c.3916_3919del)in the two infected males through NGS approach. This mutation has been further confirmed absent in 150 normal controls. We speculated this deletion mutation alters the reading framework and resulted in a PTC after new reading frame of 8 amino acids (p.Ser1306Thrfs*9).

Detailed description for clinical features is provided in Table 1. The two male patients (LZ-IV:1 and LZ-IV:2) presented bilateral congenital nuclear cataracts and both of them received cataract surgeries at an early age (Figure 2). Additional ocular features include microcornea and strabismus (Figure 2). LZ-IV:1 manifest subtle facial and dental abnormalities including a long face and narrow mandible, screwdriver-shaped incisors and diastema, while LZ-IV:2 did not show these non-ocular abnormalities. Other systemic abnormalities (cardiovascular abnormalities, mental retardation and brachymetacarpalia)were not shown in these two male patients.

NHS is a highly phenotypic heterogeneous syndrome. In this syndrome, researchers failed to find out the correlation between genotype mutation and phenotype severity

. In this study, identical non-ocular features were subtle and none of them presented mental retardation. This may be the result of allelic heterogeneity or the additional function of modifier genes

.

Targeted next-generation sequencing (NGS)approach was conducted on patients LZ-IV:1 and LZIV:2 using a previously described microarray targeting 316 ophthalmic disease relevant genes

. Library preparation,qualification, NGS with the Illumina HiSeq2000 platform(Illumina, Inc., San Diego, CA, USA), and bioinformatics analyses were performed as detailed previously

.Coverage and mean depth for NGS were calculated. All initially detected variants were subsequently filtered against five SNP databases, including dbSNP138, HapMap Project,1000 Genome Project, YH database, and Exome Variant Server. Sanger sequencing was subsequently conducted for intrafamilial cosegregation analysis and prevalence test in 150 unrelated healthy controls. Standard protocol for Sanger sequencing has been discussed previously

.

由表6說明，土樣1土壤中添加2%骨炭(A)化學(xué)修復(fù)劑時(shí)土壤中的重金屬鋅、鉛、鉻、銅、砷、鎘含量均有所下降。其中在種有狼尾草的土壤區(qū)域主要污染物鋅含量下降幅度最大，下降值為132.5mg/L。在種有狼尾草的土壤區(qū)域主要污染物砷含量下降幅度最大，最大值為2.3mg/L。

In summary, our study presented the clinical manifestations of affected males and female carriers in a Chinese family diagnosed with NHS, and identified a previously unreported frameshift mutation. The result of the present study suggested that the diagnosis of NHS or other genetic diseases require a combination of genetic analysis and clinical assessment.Besides, our result broadened the spectrum of NHS mutation and can provide genetic counselling for NHS patients.

However, our study only identified a frameshift mutation and

provided genetic evidence. Therefore, future experiments exploring the underlying mechanisms of this mutation are needed.

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