Identification of GALNT14 as a novel neuroblastoma predisposition gene

Abstract

Although several genes have been associated to neuroblastoma (NB) predisposition and aggressiveness, further genes are likely involved in the overall risk of developing this pediatric cancer. We thus carried out whole-exome sequencing on germline DNA from two affected second cousins and two unlinked healthy relatives from a large family with hereditary NB. Bioinformatics analysis revealed 6999 variations that were exclusively shared by the two familial NB cases. We then considered for further analysis all unknown or rare missense mutations, which involved 30 genes. Validation and analysis of these variants led to identify a GALNT14 mutation (c.802C > T) that properly segregated in the family and was predicted as functionally damaging by PolyPhen2 and SIFT. Screening of 8 additional NB families and 167 sporadic cases revealed this GALNT14 mutation in the tumors of two twins and in the germline of one sporadic NB patient. Moreover, a significant association between MYCN amplification and GALNT14 expression was observed in both NB patients and cell lines. Also, GALNT14 higher expression is associated with a worse OS in a public dataset of 88 NB samples (http://r2.amc.nl). GALNT14 is a member of the polypeptide N-acetylgalactosaminyl-transferase family and maps closely to ALK on 2p23.1, a region we previously discovered in linkage with NB in the family here considered. The aberrant function of GALNTs can result in altered glycoproteins that have been associated to the promotion of tumor aggressiveness in various cancers. Although rare, the recurrence of this mutation suggests GALNT14 as a novel gene potentially involved in NB predisposition.

Keywords: GALNT14; O-glycosylation; exome-sequencing; genetic predisposition; neuroblastoma.

Figure 1. Pipeline schematic

(A) conceptual workflow; (B) tools used in the present study.

Figure 2. GALNT14 c.802C > T (R268C) mutation in NB

(A) Pedigree of the NB family considered for mutation screening. All available individuals are coded with an E followed by a number. Family members analyzed by whole-exome sequencing are underlined. Germline segregation of the R268C GALNT14 mutation is reported. (B) Pedigree of the small NB family with two heterozygous twins harboring a somatic R268C GALNT14 mutation. (C) Example of the c.802C > T transition as observed by Sanger sequencing.

Figure 3. GALNT14 maps close to ALK

Representation of the relative distances of MYCN, ALK and GALNT14 on chromosome 2p.

Figure 4. GALNT14 mRNA expression in NB

Expression of GALNT14 in NB cell lines (A) and in GALNT14 positive NB patients (B) by qPCR. (C) GALNT14 average expression in MYCN amplified vs MYCN single copy NB cell lines. (D) GALNT14 average expression in NB patients with MYCN amplification, gain and MYCN single copy. F is the value of the Fisher exact test between groups. P-values are relative to the statistical difference of GALNT14 expression between MYCN amplified vs MYCN single copy groups and were calculated with the Mann-Whitney and T-Student tests in NB cell lines and patients, respectively. Y axis values are an arbitrary unit relative to the less expressed sample. A: MYCN Amplified; G: MYCN Gain; SC: MYCN Single Copy; n indicates the total number of samples for each group.

Figure 5. GALNT14 impact on NB overall survival

GALNT14 higher expression is a negative prognostic factor for OS either when considering all NB patients (A) or only MYCN not amplified cases (B) GALNT14 is a negative prognostic factor for OS in patients showing relapses or progression of the disease either when considering all NB patients (C) or MYCN not amplified cases (D). The cut off modus for GALNT14 expression to draw Kaplan-Meier curves derives from the scan setting.

Figure 6. GALNT14 impact on localized NB overall survival

GALNT14 higher expression is a negative prognostic factor for OS in NB stages 1-2-3 with no MYCN amplification. The cut off modus for GALNT14 expression to draw Kaplan-Meier curves derives from the scan setting.