Autosomal recessive diseases (ARD) are typically caused by a limited number of mutations whose identification is challenged by their low prevalence. Our purpose was to develop a novel approach allowing an efficient search for mutations causing ARD and evaluation of their pathogenicity without a control group. We developed Iterative Sequencing and Variant Screening (ISVS) approach based on iterative cycles of gene sequencing and mutation screening, and ISVS Simulator software ( http://zsibio.ii.pw.edu.pl/shiny/isvs/ ) for assessment of detected variants' significance. As shown by simulations, ISVS efficiently identifies and correctly classifies pathogenic mutations except for cases where the gene of interest has extremely high number of low frequency nonpathogenic variants. By applying ISVS, we found 4 known and 9 novel (p.C73Y, p.S124L, p.C194Mfs*17, c.782 + 2 T > A, c.953-5 A > G, p.L325Q, p.D334Mfs*24, p.R436G, p.M448T) TMPRSS3 variants among deaf patients. For 3 known and 5 novel variants the disease association was supported by ISVS Simulator odds >90:1. Pathogenicity of 6 novel mutations has been supported by in-silico predictions of variants' deleteriousness. By directly comparing variant prevalence in patients and controls, disease association was demonstrated only for two variants and it was relatively weak (P < 0.05). Summarizing, ISVS strategy and ISVS Simulator are useful for detection of genetic variants causing AR diseases.