Centre Henry Slade is the latest key player to be ruled out of England’s Six Nations opener against Scotland at Twickenham on Saturday.
Slade, 29, has not recovered from the hip injury he picked up playing for Exeter earlier this month.
England have also confirmed vice-captain Courtney Lawes will miss the Calcutta Cup game with a calf problem.
However, experienced hooker Jamie George and wing Anthony Watson have both been added to the 36-man squad.
Slade’s absence leaves Dan Kelly, Joe Marchant, Ollie Lawrence and Manu Tuilagi as the centres in the squad along with captain Owen Farrell, although the England captain is likely to be deployed at fly-half.
England are hopeful Slade’s injury is not a long-term issue, but his unavailability leaves new head coach Steve Borthwick with much to ponder in terms of his midfield.
Lawes had been expected to miss at least the Scotland game after limping off playing for Northampton last weekend and he now faces a race to be involved in the championship.
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“Courtney is a really tough loss for us, on and off the pitch,” Lawes’ Saints team-mate Lewis Ludlam told BBC Sport.
“He is obviously a fantastic player with heaps of experience. He is missed in camp, but we have however many other blokes ready to step up. There is fierce competition.”
George’s return to the squad is a boost given England’s lack of depth at hooker, although he has yet to complete the return-to-play protocols after concussion.
Watson is also a high-profile addition and is now in contention to face Scotland despite not being named in the initial Six Nations squad.
The double British and Irish Lion has been in excellent form for Leicester this season and adds experience in the back three, with Jack Nowell and Jonny May both overlooked.
Borthwick will trim down his squad on Tuesday evening before confirming his matchday 23 on Thursday afternoon.