Germ line mutations in shelterin complex genes are associated with familial chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL) can be familial; however, thus far no rare germ line disruptive alleles for CLL have been identified. We performed whole-exome sequencing of 66 CLL families, identifying 4 families where loss-of-function mutations in protection of telomeres 1 (POT1) co-segregated with CLL. The p.Tyr36Cys mutation is predicted to disrupt the interaction between POT1 and the telomeric overhang. The c.1164-1G>A splice-site, p.Gln358SerfsTer13 frameshift, and p.Gln376Arg missense mutations are likely to impact the interaction between POT1 and adrenocortical dysplasia homolog (ACD), which is a part of the telomere-capping shelterin complex. We also identified mutations in ACD (c.752-2A>C) and another shelterin component, telomeric repeat binding factor 2, interacting protein (p.Ala104Pro and p.Arg133Gln), in 3 CLL families. In a complementary analysis of 1083 cases and 5854 controls, the POT1 p.Gln376Arg variant, which has a global minor allele frequency of 0.0005, conferred a 3.61-fold increased risk of CLL (P = .009). This study further highlights telomere dysregulation as a key process in CLL development.

Figure 1

Rare POT1, ACD, and TERF2IP mutations in CLL families. Black-filled symbols indicate CLL cases, other cancers are indicated by a red-filled symbol, and an unfilled symbol indicates an individual with no known cancer. These symbols have a central dot to indicate cases that were exome sequenced. A central blue dot denotes a shelterin gene mutation carrier; a peach dot denotes a WT individual. A line through a symbol indicates that an individual is deceased. Age of diagnosis (in years) is listed for CLL cases. Splice acceptor variants are numbered relative to POT1 transcript NM_015450 and ACD transcript NM_001082486. NHL, non-Hodgkin lymphoma.

Figure 2

Impact of rare familial mutations on POT1 protein. (A) Schematic showing the position of germ line POT1 mutations identified in CLL families relative to OB domains (red) and ACD binding region (blue). Also shown are somatic POT1 mutations identified in previous studies of CLL patients^, (unshaded background) and germ line mutations found in familial cutaneous melanoma^, (peach background). (B) Cross-species conservation of POT1 amino acids subject to missense mutation in CLL families. (C) Schematic of the crystal structure of human POT1 N-terminal OB domains bound to a telomeric DNA sequence (PDB 3KJP), illustrating the proximity of tyrosine 36 to the DNA strand. OB domains are shown in gray, DNA in blue, and Tyr.36 is highlighted in magenta.

Figure 3

Impact of POT1 splice acceptor site mutation on splicing. (A) Splice acceptor site consensus scores predicted by MaxEntScan for each base from the natural POT1 intron 13/exon 14 boundary across exon 14. For clarity, only part of the intron (lower case text above black line) and exon (upper case text above black box) are shown. The predicted score for the unmutated natural splice site (red bar) is also labeled. Positive scores are otherwise marked in blue and negative scores in peach. (B) MaxEntScan splice acceptor consensus scores for the same region based upon the sequence of c.1164-1G>A POT1 mutation carriers. The scores of the mutated natural splice acceptor (pink bar) and the predicted alternative splice site with the highest MaxEntScan score (43 bp downstream) are labeled. The part of exon 14 that would be removed by use of this splice site is indicated by a gray box. (C) Abnormal splicing product detected by RT-PCR using cDNA from a CLL case (Ca) carrying the c.1164-1G>A mutation. This product is absent from control (Co) cDNA. bp, base pairs; L, ladder; NT, no template reaction.