Mutations In Cis Research Articles

Abstract 3572: Characterization of chromosomal allelic imbalances through RNA-seq

Abstract Transcriptome sequencing (mRNA-seq) is becoming a very versatile technique for profiling tumors, extending beyond its original intent of transcript quantification, identification of alternative transcripts, and detection of gene fusions. For instance, through recent advancements in RNA-seq data analyses, one can now computationally assess allele-specific gene expression and generate profiles of expressed somatic mutations. Here, we demonstrate the ability to identify chromosomal allelic imbalances (AI) through detection of haplotype-specific patterns in gene transcripts. This class of RNA-based observations may potentially reveal DNA-level chromosomal allelic imbalances or uncover large regions of transcription deregulation. From the TCGA Uterine Carcinosarcoma project, we downloaded exome and RNA sequencing data for 48 patients’ tumor/normal sample pairs in addition to their clinical annotations. We also downloaded Affymetrix SNP6-based DNA copy number event calls made by the TCGA for use as a gold standard when evaluating the AI calls in the exome and RNA-seq. AI calls were made in both the exome and RNA-seq data by: (1) calling 1000 Genomes genotypes in the sequencing data, (2) phasing haplotypes and then (3) characterizing haplotype imbalances using a tool that we developed called hapLOHseq. hapLOHseq applied to the exome and RNA-seq data both resulted in a 72% specificity for identifying the gold standard AI events. In RNA-seq data we detected 43% of the chromosomal AI events identified in the exome sequencing data. When considering AI events specifically detected in the RNA-seq and not the gold standard (RNA-specific AI), the data suggest that higher RNA-specific AI loads could be negatively associated with survival (p-val = 0.076), with higher RNA-specific AI load patients having a median survival of 771 days compared to 1526 days for those patients with lower loads of RNA-specific AI. In conclusion, our results suggest that analysis of chromosomal AI in RNA-seq has equal specificity for detecting DNA-level AI when compared to exome sequencing, although at lower sensitivities. Clinically, our analyses suggest that patients with higher RNA-specific AI load may have a worse overall survival prognosis. The AI we are identifying in the RNA-seq samples may reflect large-scale transcription defects, resulting in a negative impact on the survival of patients. One possible cause of RNA-specific allelic imbalance could be the presence of cis mutations that impact a large-region of the transcription of one of the two haplotypes. Currently, we are identifying areas for improvement in our analytical methods, while interrogating and characterizing exome and RNA-seq AI in additional data sets. Citation Format: Francis A. San Lucas, Yihua Liu, Zachary Weber, Erik Ehli, Gareth Davies, Paul Scheet. Characterization of chromosomal allelic imbalances through RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3572. doi:10.1158/1538-7445.AM2017-3572

Alport syndrome: impact of digenic inheritance in patients management.

Alport syndrome (ATS) is a genetically heterogeneous nephropathy with considerable phenotypic variability and different transmission patterns, including monogenic (X-linked/autosomal) and digenic inheritance (DI). Here we present a new series of families with DI and we discuss the consequences for genetic counseling and risk assessment. Out of five families harboring variants in more than one COL4 gene detected by next generation sequencing (NGS), minigene-splicing assay allowed us to identify four as true digenic. Two families showed COL4A3/A4 mutations in cis, mimicking an autosomal dominant inheritance with a more severe phenotype and one showed COL4A3/A4 mutations in trans, mimicking an autosomal recessive inheritance with a less severe phenotype. In a fourth family, a de novo mutation (COL4A5) combined with an inherited mutation (COL4A3) triggered a more severe phenotype. A fifth family, predicted digenic on the basis of silico tools, rather showed monogenic X-linked inheritance due to a hypomorphic mutation, in accordance with a milder phenotype. In conclusion, this study highlights the impact of DI in ATS and explains the associated atypical presentations. More complex inheritance should be therefore considered when reviewing prognosis and recurrence risks. On the other side, these findings emphasize the importance to accompany NGS with splicing assays in order to avoid erroneous identification of at risk members.

EGFR L858M/L861Q cis Mutations Confer Selective Sensitivity to Afatinib

IntroductionTyrosine kinase inhibitors (TKIs) have been developed to treat patients with EGFR-mutant lung cancers. However, the therapeutic efficacy of TKIs in patients with uncommon EGFR mutations remains unclear. MethodsNext-generation sequencing was performed on a patient’s lung adenocarcinoma tumor sample, revealing rare combined in cis (on the same allele) EGFR mutations. Stable Ba/F3 and NIH-3T3 cell lines harboring the mutations were established to investigate the effect of first-, second-, and third-generation EGFR TKIs on cell proliferation by MTS assay and EGFR phosphorylation by Western blotting. ResultsEGFR L858M/L861Q mutations in cis were detected in the tumor of a patient with NSCLC. The patient demonstrated primary resistance to erlotinib and was subsequently treated with afatinib, which caused tumor regression. In in vitro studies, first- and third-generation TKIs exhibited a decreased capacity to prevent EGFR phosphorylation and inhibit cell proliferation in EGFR L858M/L861Q cells compared with cells harboring the common EGFR L858R point mutation. In contrast, afatinib treatment reduced proliferation and inhibited EGFR phosphorylation in L858M/L861Q- and L858R-mutant cells at similar concentrations. ConclusionsAfatinib may be a beneficial therapeutic option for a subset of patients with lung cancer who harbor rare EGFR mutations in their tumors. Understanding how uncommon mutations affect protein structure and TKI binding will be important for identifying effective targeted therapies for these patients.

Double Jeopardy: The Rubber Ball Bounces Twice

Soblet et al. describe cis mutations in TEK/Tie-2 in blue rubber bleb nevus and sporadic vascular malformations. This suggests that the remaining normal allele is required for the phenotype. Second, it suggests therapeutic approaches to treatment signal transduction inhibition.

The novel complex allele [A238V;F508del] of the CFTR gene: clinical phenotype and possible implications for cystic fibrosis etiological therapies.

Few mutations in cis have been annotated for F508del homozygous patients. Southern Italy patients who at a first analysis appeared homozygous for the F508del mutation (n=63) or compound heterozygous for the F508del and another mutation in the cystic fibrosis transmembrane conductance regulator gene (n=155) were searched for the A238V mutation in exon 6. The allelic frequency of the complex allele [A238V;F508del] was 0.04. When the whole data set was used (comprised also of 56 F508del/F508del and 34 F508del/other mutation controls), no differences reached the statistical significance in the clinical parameters, except chloride concentrations which were lower in [A238V;F508del]/other mutation compared with F508del/other mutation (P=0.03). The two study groups presented less complications than the control groups. Within the minimal data set (34 F508del/F508del, 27 F508del/other mutation, 4 [A238V;F508del]/F508del cases and 5 [A238V;F508del]/other mutation cases); that is, presenting all the variables in each patient, forced expiratory volume in 1 s and forced vital capacity presented a trend to lower levels in the study groups in comparison with the F508del/F508del group, and C-reactive protein approximated statistically significant higher levels in the [A238V;F508del]/other mutation as compared with F508del/F508del patients (P=0.09). The analysis of statistical dependence among the variables showed a significant anticorrelation between chloride and body mass index in the [A238V;F508del]/other mutation group. In conclusion, the complex allele [A238V;F508del] seems to be associated with less general complications than in the control groups, on the other hand possibly giving a worse pulmonary phenotype and higher systemic/local inflammatory response. These findings have implications for the correct recruitment and clinical response of F508del patients in the clinical trials testing the new etiological drugs for cystic fibrosis.

Compound mutations in BCR-ABL1 are not major drivers of primary or secondary resistance to ponatinib in CP-CML patients

AbstractBCR-ABL1 kinase domain mutations can confer resistance to first- and second-generation tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). In preclinical studies, clinically achievable concentrations of the third-generation BCR-ABL1 TKI ponatinib inhibit T315I and all other single BCR-ABL1 mutants except T315M, which generates a single amino acid exchange, but requires 2 sequential nucleotide exchanges. In addition, certain compound mutants (containing ≥2 mutations in cis) confer resistance. Initial analyses based largely on conventional Sanger sequencing (SS) have suggested that the preclinical relationship between BCR-ABL1 mutation status and ponatinib efficacy is generally recapitulated in patients receiving therapy. Thus far, however, such analyses have been limited by the inability of SS to definitively identify compound mutations or mutations representing less than ∼20% of total alleles (referred to as “low-level mutations”), as well as limited patient follow-up. Here we used next-generation sequencing (NGS) to define the baseline BCR-ABL1 mutation status of 267 heavily pretreated chronic phase (CP)-CML patients from the PACE trial, and used SS to identify clonally dominant mutants that may have developed on ponatinib therapy (30.1 months median follow-up). Durable cytogenetic and molecular responses were observed irrespective of baseline mutation status and included patients with compound mutations. No single or compound mutation was identified that consistently conferred primary and/or secondary resistance to ponatinib in CP-CML patients. Ponatinib is effective in CP-CML irrespective of baseline mutation status.

MG-125 CYP21A2 mutation spectrum in congenital adrenal hyperplasia identified from molecular genetic testing

Background Mutations in CYP21A2, the gene encoding 21-hydroxylase, are identified in >80% of individuals with congenital adrenal hyperplasia (CAH). CYP21A2 and its highly homologous pseudogene, CYP21A1P , reside in close proximity; as a result >90% of CYP21A2 mutation-containing alleles are caused by gene conversion from CYP21A1P or CYP21A2 deletions arising from non-allelic homologous recombination. Further, CYP21A2 duplications exist and provide a source of false negative and false positive results as carriers of a CYP21A2 deletion may be masked by a CYP21A2 duplication on the opposite allele (2+0 configuration) or carriers of a point mutation may also carry a second non-mutated CYP21A2 in cis. Objectives The purpose of this study was to investigate the distribution of CYP21A2 mutations identified from molecular genetic tested CAH individuals and determine the frequency of 2+0 carriers in a control population. Methods A total of 469 CAH individuals were screened for CYP21A2 mutation by MLPA and common mutation and/or sequencing analysis. A PCR-based assay was developed and to detect 2+0 CYP21A2 deletion carriers. Results The p. V282L, c.293–13A/C >G, and CYP21A2 gene deletion were the most frequent mutations identified in CAH. Cases with two independent mutations in cis and mutation-containing CYP21A2 duplications were observed. No silent 2+0 CYP21A2 deletion carriers were identified in our control population. Conclusions Segregation and copy number analysis are critical in CYP21A2 testing. A PCR-based assay can be used to detect CYP21A2 duplications, although the 2+0 silent carrier frequency is rare.

Non-syndromic hearing loss caused by the dominant cis mutation R75Q with the recessive mutation V37I of the GJB2 (Connexin 26) gene.

GJB2 alleles containing two cis mutations have been rarely found in non-syndromic hearing loss. Herein, we present a Korean patient with non-syndromic hearing loss caused by the R75Q cis mutation with V37I, which arose de novo in the father and was inherited by the patient. Biochemical coupling and hemichannel permeability assays were performed after molecular cloning and transfection of HEK293T cells. Student's t-tests or analysis of variance followed by Tukey's multiple comparison test was used as statistical analysis. Biochemical coupling was significantly reduced in connexin 26 (Cx26)-R75Q- and Cx26-V37I-transfected cells, with greater extent in Cx26-R75Q and Cx26-R75Q+V37I cells. Interestingly, our patient and his father with the mutations had more residual hearing compared with patients with the dominant mutation alone. Although the difference in hemichannel activity between R75Q alone and R75Q in combination with V37I failed to reach significance, it is of note that there is a possibility that V37I located upstream of R75Q might have the ability to ameliorate R75Q expression. Our study emphasizes the importance of cis mutations with R75Q, as the gene effect of R75Q can be modulated depending on the type of additional mutation.

N1303K (c.3909C>G) mutation and splicing: implication of its c.[744-33GATT(6); 869+11C>T] complex allele in CFTR exon 7 aberrant splicing.

Cystic Fibrosis is the most common recessive autosomal rare disease found in Caucasians. It is caused by mutations on the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that encodes a protein located on the apical membrane of epithelial cells. c.3909C>G (p.Asn1303Lys, old nomenclature: N1303K) is one of the most common worldwide mutations. This mutation has been found at high frequencies in the Mediterranean countries with the highest frequency in the Lebanese population. Therefore, on the genetic level, we conducted a complete CFTR gene screening on c.3909C>G Lebanese patients. The complex allele c.[744-33GATT(6); 869+11C>T] was always associated with the c.3909C>G mutation in cis in the Lebanese population. In cellulo splicing studies, realized by hybrid minigene constructs, revealed no impact of the c.3909C>G mutation on the splicing process, whereas the associated complex allele induces minor exon skipping.

Genetic evidence of a precisely tuned dysregulation in the hypoxia signaling pathway during oncogenesis.

The classic model of tumor suppression implies that malignant transformation requires full "two-hit" inactivation of a tumor-suppressor gene. However, more recent work in mice has led to the proposal of a "continuum" model that involves more fluid concepts such as gene dosage-sensitivity and tissue specificity. Mutations in the tumor-suppressor gene von Hippel-Lindau (VHL) are associated with a complex spectrum of conditions. Homozygotes or compound heterozygotes for the R200W germline mutation in VHL have Chuvash polycythemia, whereas heterozygous carriers are free of disease. Individuals with classic, heterozygous VHL mutations have VHL disease and are at high risk of multiple tumors (e.g., CNS hemangioblastomas, pheochromocytoma, and renal cell carcinoma). We report here an atypical family bearing two VHL gene mutations in cis (R200W and R161Q), together with phenotypic analysis, structural modeling, functional, and transcriptomic studies of these mutants in comparison with classical mutants involved in the different VHL phenotypes. We demonstrate that the complex pattern of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of VHL protein (pVHL) dysfunction in hypoxia signaling pathways. Thus, by studying naturally occurring familial mutations, our work validates in humans the "continuum" model of tumor suppression.

Mutation in the novel nuclear-encoded mitochondrial protein CHCHD10 in a family with autosomal dominant mitochondrial myopathy.

Mitochondrial myopathies belong to a larger group of systemic diseases caused by morphological or biochemical abnormalities of mitochondria. Mitochondrial disorders can be caused by mutations in either the mitochondrial or nuclear genome. Only 5% of all mitochondrial disorders are autosomal dominant. We analyzed DNA from members of the previously reported Puerto Rican kindred with an autosomal dominant mitochondrial myopathy (Heimann-Patterson et al. 1997). Linkage analysis suggested a putative locus on the pericentric region of the long arm of chromosome 22 (22q11). Using the tools of integrative genomics, we established chromosome 22 open reading frame 16 (C22orf16) (later designated as CHCHD10) as the only high-scoring mitochondrial candidate gene in our minimal candidate region. Sequence analysis revealed a double-missense mutation (R15S and G58R) in cis in CHCHD10 which encodes a coiled coil-helix-coiled coil-helix protein of unknown function. These two mutations completely co-segregated with the disease phenotype and were absent in 1,481 Caucasian and 80 Hispanic (including 32 Puerto Rican) controls. Expression profiling showed that CHCHD10 is enriched in skeletal muscle. Mitochondrial localization of the CHCHD10 protein was confirmed using immunofluorescence in cells expressing either wild-type or mutant CHCHD10. We found that the expression of the G58R, but not the R15S, mutation induced mitochondrial fragmentation. Our findings identify a novel gene causing mitochondrial myopathy, thereby expanding the spectrum of mitochondrial myopathies caused by nuclear genes. Our findings also suggest a role for CHCHD10 in the morphologic remodeling of the mitochondria.

The expansion of body coloration involves coordinated evolution in cis and trans within the pigmentation regulatory network of Drosophila prostipennis

The generation of complex morphological features requires the precisely orchestrated expression of numerous genes during development. While several traits have been resolved to evolutionary changes within a single gene, the evolutionary path by which genes derive co-localized or mutually excluded expression patterns is currently a mystery. Here we investigate how the Drosophila pigmentation gene network was altered in Drosophila prostipennis, a species in the Drosophila melanogaster subgroup, that evolved expanded abdominal pigmentation. We show that this expansion involved broadened expression of the melanin-promoting enzyme genes tan and yellow, and a reciprocal withdrawn pattern of the melanin-suppressing enzyme gene ebony. To examine whether these coordinated changes to the network were generated through mutations in the cis-regulatory elements (CREs) of these genes, we cloned and tested CREs of D. prostipennis tan, ebony, and yellow in transgenic reporter assays. Regulatory regions of both tan and ebony failed to recapitulate the derived D. prostipennis expression phenotype, implicating the modification of a factor or factors upstream of both genes. However, the D. prostipennis yellow cis-regulatory region recapitulated the expanded expression pattern observed in this species, implicating causative mutations in cis to yellow. Our results provide an example in which a coordinated expression program evolved through independent changes at multiple loci, rather than through changes to a single “master regulator” directing a suite of downstream target genes. This implies a complex network structure in which each gene may be subject to a unique set of inputs, and resultantly may require individualized evolutionary paths to yield correlated gene expression patterns.

Direct involvement of the CreA transcription factor in penicillin biosynthesis and expression of the pcbAB gene in Penicillium chrysogenum.

The transcription factor CreA is the main regulator responsible for carbon repression in filamentous fungi. CreA is a wide domain regulator that binds to regulatory elements in the promoters of target genes to repress their transcription. Penicillin biosynthesis and the expression of penicillin biosynthetic genes are subject to carbon repression. However, evidence of the participation of CreA in this regulation is still lacking, and previous studies on the promoter of the pcbC gene of Aspergillus nidulans indicated the lack of involvement of CreA in its regulation. Here we present clear evidence of the participation of CreA in carbon repression of penicillin biosynthesis and expression of the pcbAB gene, encoding the first enzyme of the pathway, in Penicillium chrysogenum. Mutations in cis of some of the putative CreA binding sites present in the pcbAB gene promoter fused to a reporter gene caused an important increase in the measured enzyme activity in glucose-containing medium, whereas activity in the medium with lactose was not affected. An RNAi strategy was used to attenuate the expression of the creA gene. Transformants expressing a small interfering RNA for creA showed higher penicillin production, and this increase was more evident when glucose was used as carbon source. These results confirm that CreA plays an important role in the regulation of penicillin biosynthesis in P. chrysogenum and opens the possibility of its utilization to improve the industrial production of this antibiotic.

What the X Has to Do with It: Differences in Regulatory Variability between the Sexes in Drosophila simulans

The mechanistic basis of regulatory variation and the prevailing evolutionary forces shaping that variation are known to differ between sexes and between chromosomes. Regulatory variation of gene expression can be due to functional changes within a gene itself (cis) or in other genes elsewhere in the genome (trans). The evolutionary properties of cis mutations are expected to differ from mutations affecting gene expression in trans. We analyze allele-specific expression across a set of X substitution lines in intact adult Drosophila simulans to evaluate whether regulatory variation differs for cis and trans, for males and females, and for X-linked and autosomal genes. Regulatory variation is common (56% of genes), and patterns of variation within D. simulans are consistent with previous observations in Drosophila that there is more cis than trans variation within species (47% vs. 25%, respectively). The relationship between sex-bias and sex-limited variation is remarkably consistent across sexes. However, there are differences between cis and trans effects: cis variants show evidence of purifying selection in the sex toward which expression is biased, while trans variants do not. For female-biased genes, the X is depleted for trans variation in a manner consistent with a female-dominated selection regime on the X. Surprisingly, there is no evidence for depletion of trans variation for male-biased genes on X. This is evidence for regulatory feminization of the X, trans-acting factors controlling male-biased genes are more likely to be found on the autosomes than those controlling female-biased genes.

Bax 2 Family Alternative Splicing Salvages Bax Microsatellite-Frameshift Mutations

Mutation or aberrant splicing can interrupt gene expression. Tumor suppressor Bax is one of the susceptible genes prone to microsatellite frameshifting mutations in coding regions. As a result, tumors exhibiting microsatellite instability (MSI) often present a "Bax-negative" phenotype. We previously reported that some Bax-negative cells in fact contain a functional Bax isoform (BaxΔ2), generated when unique alternative splicing "salvages" the shifted reading frame introduced by a microsatellite mutation. Here we compared Bax alternative splicing profiles in a range of cell lines and primary tumors with and without Bax microsatellite mutations. We found that MSI tumors exhibit a high Bax alternative splicing frequency, especially in exon 2, and produce a family of alternatively spliced isoforms that retain many important Bax functional domains. Surprisingly, these BaxΔ2 family isoforms can rescue Bax from all common microsatellite frameshift mutations. Production of BaxΔ2 requires specific cis mutations, while trans components are not cell-type specific. Furthermore, all BaxΔ2 family isoforms are more potent cell death inducers than the parental Bax without directly targeting mitochondria. These results indicate that the BaxΔ2 family can potentially salvage Bax tumor suppressor expression otherwise lost to mutation.

Functional analysis of 11 novel GBA alleles

Gaucher disease is the most frequent lysosomal storage disorder due to the deficiency of the acid β-glucosidase, encoded by the GBA gene. In this study, we report the structural and functional characterization of 11 novel GBA alleles. Seven single missense alleles, P159S, N188I, E235K, P245T, W312S, S366R and W381C, and two alleles carrying in cis mutations, (N188S; G265R) and (E326K; D380N), were studied for enzyme activity in transiently transfected cells. All mutants were inactive except the P159S, which retained 15% of wild-type activity. To further characterize the alleles carrying two in cis mutations, we expressed constructs bearing singly each mutation. The presence of G265R or D380N mutations completely abolished enzyme activity, while N188S and E326K mutants retained 25 and 54% of wild-type activity, respectively. Two mutations, affecting the acceptor splice site of introns 5 (c.589-1G>A) and 9 (c.1389-1G>A), led to the synthesis of aberrant mRNA. Unpredictably, family studies showed that two alleles resulted from germline or 'de novo' mutations. These results strengthen the importance of performing a complete and accurate molecular analysis of the GBA gene in order to avoid misleading conclusions and provide a comprehensive functional analysis of new GBA mutations.

Two Rare Hemoglobin Variants with α Thalassemia in Eastern Indian Population

The current work focuses on two rare hemoglobin (Hb) variants - Hb Grange-Blanche and Hb Hofu - found for the first time in association with α-thalassemia in Eastern India. The unusual case of Hb Grange-Blanche and FS 41/42(-CTTT) mutations in cis throws light on importance of multiple mutations and its coinheritance with αααanti3.7 triplication indicates a possible cause for the clinical severity in β-thalassemia carriers.

EGFR L858R Mutation May Go Undetected Because of P848L in cis Mutation

<i>EGFR</i> L858R Mutation May Go Undetected Because of P848L in <i>cis</i> Mutation

Novel CFTR Variants Identified during the First 3 Years of Cystic Fibrosis Newborn Screening in California

California uses a unique method to screen newborns for cystic fibrosis (CF) that includes gene scanning and DNA sequencing after only one California-40 cystic fibrosis transmembrane conductance regulator (CFTR) panel mutation has been identified in hypertrypsinogenemic specimens. Newborns found by sequencing to have one or more additional mutations or variants (including novel variants) in the CFTR gene are systematically followed, allowing for prospective assessment of the pathogenic potential of these variants. During the first 3 years of screening, 55 novel variants were identified. Six of these novel variants were discovered infive screen-negative participants and three were identified in multiple unrelated participants. Ten novelvariants (c.2554_2555insT, p.F1107L, c.-152G>C, p.L323P, p.L32M, c.2883_2886dupGTCA, c.2349_2350insT, p.K114del, c.-602A>T, and c.2822delT) were associated with a CF phenotype (42% of participants were diagnosed at 4 to 25 months of age), whereas 26 were associated with CFTR-related metabolic syndrome to date. Associations with the remaining novel variants were confounded by the presence of other diseases or other mutations in cis or by inadequate follow-up. These findings have implications for how CF newborn screening and follow-up is conducted and will help guide which genotypes should, and which should not, be considered screen positive for CF in California and elsewhere.

Identification of a SNP in a Regulatory Region of GJB2 Associated With Idiopathic Nonsyndromic Autosomal Recessive Hearing Loss in a Multicenter Study

Additional genetic changes in the regulatory region of the human GJB2 gene encoding the gap junction protein (Connexin 26) may contribute to sensorineural hearing loss. Mutations in GJB2 cause up to 50% of autosomal recessive nonsyndromic hearing impairment (NSHI). In the present study, we screened the putative 5' GJB2 regulatory region for novel alterations. In idiopathic familial cases of NSHI lacking known pathogenic alterations in GJB2, we identified a T→C transition (refSNP: rs117685390) in a putative transcription factor binding sequence 228 bp proximal to the transcriptional start site at a homozygous frequency of 0.125 (n = 40), significantly overrepresented in comparison to the homozygous allele frequencies of 0.043 in the normal-hearing Caucasian population (n = 211; p < 0.001). In a NSHI family, inheritance of the rs117685390 C allele segregated on independent chromosomes with NSHI in conjunction with heterozygous inheritance of c.35delG, the most common Caucasian mutation in the GJB2 coding region. In a patient group (n = 32) bearing heterozygous pathogenic c.35delG mutations, - rs117685390 C allele homozygosity was also highly overrepresented (0.25; p < 0.001) and not exclusively linked to the c.35delG mutation in cis in patients homozygous for c.35delG. However, in the majority of NSHI homozygous c.35delG chromosomes examined (91/94), c.35delG homozygosity was linked to the rs117685390 C allele in cis. These results suggest that the rs117685390 C allele could represent a biomarker for the development of NSHI in Caucasian populations and may be included in risk assessment for the development of NSHI.