You don't show what's in file, but why not just use another option variable? (Note, this is untested.)

my %help_options = (
   "color!" => sub { $options{USE_COLOR_CMDLINE} = $_[1] },
   "conf=s" => sub { $options{CONF} = $_[1] },
);
GetOptions( %help_options );

do  $options{CONF} if defined $options{CONF};

# Override it if specified on the command line.
$options{USE_COLOR} = $options{USE_COLOR_CMDLINE}
    if exists $options{USE_COLOR_CMDLINE};
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That's not necessarily the easiest or best way to accomplish it, but it's probably the smallest change to your example code that would do it.

On a separate note, I cringe at the idea that your program variables are spread among its own and the external file. (In other words, the external file knows the name of the %options hash and the particular key values, so if you change one you have to change the other.) I'd much rather use a "ini" style file with var = value pairs or some such, where I can map the keys to my variables through a hash or something. Of course, I don't know what limitations you're working under, so maybe this isn't practical for you, but if it is, I'd really recommend the change.