The defectors, who include a minister and a vice minister of Berlusconi's centre-right coalition government, have called their new grouping Future and Freedom for Italy.
The Berlusconi government's majority had comprised 271 MPs of the PDL, 59 from the Northern League coalition partner and 12 other centre-right deputies.
The opposition has 269 seats, while 19 lawmakers oscillate between the two camps.
The minimum number of seats for an absolute majority is 316 out of the total 630.
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