Thanks to the advent of rapid DNA sequencing technology and its prevalence, many disease-associated genetic variants are rapidly identified in many genes from patient samples. However, the subsequent effort to experimentally validate and define their pathological roles is extremely slow. Consequently, the pathogenicity of most disease-associated genetic variants is solely speculated in silico, which is no longer deemed compelling. We developed an experimental approach to efficiently quantify the pathogenic effects of disease-associated genetic variants with a focus on SLC26A4, which is essential for normal inner ear function. Alterations of this gene are associated with both syndromic and nonsyndromic hereditary hearing loss with various degrees of severity. We established HEK293T-based stable cell lines that express pendrin missense variants in a doxycycline-dependent manner, and systematically determined their anion transport activities with high accuracy in a 96-well plate format using a high throughput plate reader. Our doxycycline dosagedependent transport assay objectively distinguishes missense variants that indeed impair the function of pendrin from those that do not (functional variants). We also found that some of these putative missense variants disrupt normal messenger RNA splicing. Our comprehensive experimental approach helps determine the pathogenicity of each pendrin variant, which should guide future efforts to benefit patients. K E Y W O R D SDFNB4, hereditary hearing loss, Pendred syndrome, pendrin, SLC26A4

Cadherin-related 23 (CDH23) is an adhesive protein important for hearing and vision, while CAMSAP3/Marshalin is a microtubule (MT) minus-end binding protein that regulates MT networks. Although both CDH23 and CAMSAP3/Marshalin are expressed in the organ of Corti, and carry several protein-protein interaction domains, no functional connection between these two proteins has been proposed. In this report, we demonstrate that the C isoform of CDH23 (CDH23-C) directly binds to CAMSAP3/Marshalin and modifies its function by inhibiting CAMSAP3/Marshalin-induced bundle formation, a process that requires a tubulin-binding domain called CKK. We further identified a conserved N-terminal region of CDH23-C that binds to the CKK domain. This CKK binding motif (CBM) is adjacent to the domain that interacts with harmonin, a binding partner of CDH23 implicated in deafness. Because the human Usher Syndrome 1D-associated mutation, CDH23 R3175H, maps to the CBM, we created a matched mutation in mouse CDH23-C at R55H. Both in vivo and in vitro assays decreased the ability of CDH23-C to interact with CAMSAP3/Marshalin, indicating that the interaction between CDH23 and CAMSAP3/Marshalin plays a vital role in hearing and vision. Together, our data suggest that CDH23-C is a CAMSAP3/Marshalin-binding protein that can modify MT networks indirectly through its interaction with CAMSAP3/Marshalin.

Neuropathic pain is a challenge for clinicians because it is resistant to commonly prescribed analgesics, such as opioids and nonsteroidal antiinflammatory drugs. Fortunately, adjuvant analgesics, drugs not typically thought of as pain relievers, may be effective. It is helpful to classify adjuvant analgesics used to treat neuropathic pain into two broad categories: (1) membrane stabilizing agents, which inhibit ectopic discharges on damaged neural membranes, and (2) drugs that enhance dorsal horn inhibition, which may augment biogenic amine or GABAergic mechanisms in the dorsal horn of the spinal cord. Current evidence regarding efficacy generally does not support the use of one drug over another, and selection of a particular drug may depend on experience or expected side effects. The overall efficacy of tricyclic antidepressants for neuropathic pain is modest, and they may produce intolerable side effects. Based on current studies, gabapentin is a reasonable alternative to antidepressants, as initial monotherapy or add-on treatment, particularly for painful diabetic peripheral neuropathy and postherpetic neuralgia. From a practical standpoint, to optimize analgesia more than one drug may be necessary. Although polypharmacy is the result, this approach may improve therapy and minimize side effects. From a safety standpoint, medications generally should be started at low doses and titrated to effect. Although labor-intensive, this strategy can improve compliance and optimize patient care.