Paired, 76 base pair (bp) reads from postenrichment shotgun libraries were aligned to the reference genome. On average, 3.1 gigabases (Gb) of mapped sequence was generated per individual and 85% of reads were mapped. The average coverage of each exome was 70-fold. On average, 57,750 SNPs were called per individual (range 53,417–62,098), of which 64% were already annotated in a public database (dbSNP v131). A total of 19,743 SNPs were in common among all 4 patients and 7,701 of these were novel. A total of 396 SNPs were predicted to be damaging (loss of function) by PolyPhen2, of which 240 had a dbSNP ID. On average, 5,038 indels were called per individual, of which 13.5% were previously annotated in dbSNP v131. A total of 1,840 indels were in common among all 4 patients, 1,562 were novel (i.e., not present in dbSNP), and 367 were exonic. In order to refine our list of variants, we included only those variants that were frameshift indels or PolyPhen2 predicted to be loss of function (damaging) and were common to all 4 affected individuals and present in regions that showed evidence for linkage under a “rare variant” model of multipoint linkage analyses (; ).
Number of “probably” damaging variants identified in regions of linkage
A single SNP in the TYK2
gene on chromosome 19p13 was the only variant identified using these filter conditions (; ). The variant encodes a missense mutation in exon 3 of TYK2
that changes an alanine to threonine (A53T) and it is predicted to impact protein function by PolyPhen2 and SIFT.15
The genomic evolutionary rate profiling (GERP) score was +4.20. The GERP scores for rs897738 and rs6427384, the 2 “possibly” damaging variants, were +2.68 and −1.62, respectively (). The A53T residue is highly conserved between species.
The variant at TYK2 has previously been reported once in dbSNP by a study on cancer genomes and has an ID of rs55762744. The SNP was not reported by the 1000 Genomes project. MS has previously been shown to be associated with another, nonsynonymous SNP (rs34536443) in TYK2 (P1104A), which lies over 25 kb downstream of rs55762744. The 4 individuals sequenced did not carry the rs34536443 MS-associated SNP. There were no obvious differences with MS phenotype or HLA genotype in the TYK2 A53T variant carriers and noncarriers.
The variant was genotyped in the remaining affected family members. Of the affected individuals with DNA available, a total of 10/14 (72%) were positive for the TYK2 mutation. The unaffected family members were also genotyped and a total of 28/60 (47%) were positive for the variant. When a parent was a heterozygous carrier for the mutation, the parent transmitted to an affected offspring 10 times and did not transmit 2 times (χ2 = 5.33, p = 0.02). We then genotyped 2,104 MS “trio” families and 16 cases were positive for the TYK2 variant (0.8%). Twenty-one unaffected parents were heterozygous for rs55762744 and the risk allele was transmitted preferentially to 16 cases and not transmitted to 5 cases (χ2 = 5.76, p = 0.016). A control sample was genotyped for the variant and 10 individuals of 1,543 (0.6%) were positive for the T allele and not significantly different from the affected allele frequency.
We also tested for any rare variants in the other known MS susceptibility genes outside of the linkage regions identified by the MS GWA studies. These genes included IL2RA, IL7R, CLEC16A, CD58, TNFRSF1A, IRF8, EV15, KIF1B, KIF21B, CD40, STAT3, CBLB, CD6, CD226, and GPC5. There were no frameshift insertions, deletions, or damaging SNPs identified in any of the 4 sequenced individuals at these genes.