AIFM1 variations, as well as the phenotype progression of these patients, exploring the potential molecular pathogenic mechanism of AN. A total of 36 families of individuals with AN (50 cases) with AIFM1 variations were recruited and identified by Sanger sequencing or next-generation sequencing; the participants included 30 patients from 16 reported families and 20 new cases. We found that AIFM1-positive cases accounted for 18.6% of late-onset AN cases. Of the 50 AN patients with AIFM1 variants, 45 were male and 5 were female. The hotspot variation of this gene was p.Leu344Phe, accounting for 36.1%. A total of 19 AIFM1 variants were reported in this study, including 7 novel ones. A follow-up study was performed on 30 previously reported AIFM1-positive subjects, 16 follow-up cases (53.3%) were included in this study, and follow-up periods were recorded from 1 to 23 years with average 9.75 ± 9.89 years. There was no hearing threshold increase during the short-term follow-up period (1-10 years), but the low-frequency and high-frequency hearing thresholds showed a significant increase with the prolongation of follow-up time. The speech discrimination score progressed gradually and significantly along with the course of the disease and showed a more serious decline, which was disproportionately worse than the pure tone threshold. In addition to the X-linked recessive inheritance pattern, the X-linked dominant inheritance pattern is also observed in AIFM1-related AN and affects females. In conclusion, we confirmed that AIFM1 is the primary related gene among late-onset AN cases, and the most common recurrent variant is p.Leu344Phe. Except for the X-linked recessive inheritance pattern, the X-linked dominant inheritance pattern is another probability of AIFM1-related AN, with females affected. Phenotypical features of AIFM1-related AN suggested that auditory dyssynchrony progressively worsened over time.To decipher the genotype-phenotype correlation of auditory neuropathy (AN) caused by  Auditory neuropathy (AN) is a special type of sensorineural hearing loss with a main manifestation of impaired speech comprehension, accounting for 1.2-10% of cases of hearing loss, depending on the population .AIFM1 as the gene responsible for this kind of AN using whole exome sequencing technology in 2015 [The pathogenic mechanism of AN is currently unclear, and genetic factors may account for up to 40% of the pathogenesis of AN  and then in 2015 . Apoptos in 2015 . This prSince AN was first identified more than 20 years ago, diagnosis, particularly precision diagnosis with lesion site identification, remains a challenge. Cases with genetic basis and the identification of the genes may be helpful for the lesion site identification, deciphering the underlying mechanism of AN –9. ExcepAIFM1-positive cases in AN cases. In this study, we further identified another 20 AN cases with AIFM1 variants, including 7 novel variants and one hotspot variant, showing that the proportion of AN caused by AIFM1 in Chinese patients with delayed-onset AN was as high as 18.6% (36/194), higher than the 15.53% (16/103) observed in the previous study [AIFM1 gene pathogenic mutations were carried out, including the clinical hearing vestibular test, comprehensive clinical follow-up study data, and an in-depth exploration of the clinical characteristics of the disease and the related pathogenesis, to explore the characteristics of AIMF1 gene-positive AN, laying the theoretical basis of AN classification diagnosis. The X-linked dominant inheritance pattern is also firstly observed in AIFM1-related AN and affects females in the study as well.Up to date, there is no frequency data of The study was approved by the Committee of Medical Ethics of Chinese PLA General Hospital. Written informed consent was obtained from all participants.AIFM1 mutations who were diagnosed with AN in the Chinese PLA Institute of Otolaryngology, Chinese PLA General Hospital, from April 1997 to June 2019 were recruited for this study. The diagnostic criteria were as follows: The typical audiological characteristics were that the auditory brainstem response (ABR) had no obvious differentiation waveform or severe abnormality and that the otoacoustic emission (OAE) and/or the cochlear microphonic (CM) potential could be normally extracted. Personal or family medical evidence of hearing loss, tinnitus, vestibular symptoms, and other clinical abnormalities of both the affected members and the unaffected members of these families was identified. Pure tone threshold (PTA), speech discrimination score (SDS), ABR, OAE, CM, and electrocochleography (ECochG) were carried out as otological examination batteries to evaluate auditory status. In general, the low frequencies were primarily affected; thus, we focused on the low-frequency data and calculated PTA as the average of the thresholds of 250-1000 Hz to avoid bias in the assessment of the degree of AN hearing loss. Vestibular function evaluation included vestibular evoked myogenic potentials, oculomotor function tests, positional nystagmus tests, positioning nystagmus tests, and bithermal caloric tests. High-resolution computed tomography (CT) scans of the temporal bone and cerebral magnetic resonance imaging (MRI) were performed to exclude other possible neuropathic or anatomical disorders.A total of 50 patients with Next-generation sequencing and Sanger sequencing were performed on the patients as previously described. Variation interpretation  was based on the standards and guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG and AMP) .http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA) and R software (https://www.R-project.org), as well as G. Comparisons of the two sets of data were performed using an independent sample t-test. The comparisons of multiple sets of data were performed using one-way ANOVA. p < 0.05 represented a significant difference. Spline smoothing was performed using GAMM  to explore the change in pure tone threshold with the length of follow-up time.Statistical analysis was performed using SPSS 19.0 statistical software, Empower software  (AIFM1-positive patients ranged from 5 to 20 years (13.4 ± 3.9). In addition, the earlier the onset, the more severe the hearing impairment. The pure tone averages  of the <12-year-old group, 12- to 16-year-old group, and >16-year-old group were 55.7, 51.2, and 47.7 dB HL, respectively. The most severe hearing loss was observed in the youngest age group. However, the differences in hearing loss between the three onset age groups were not statistically significant  . The onsnificant .AIFM1 variations, seven novel and three reported variations were found, located in the FAD, NADH, and C-terminus. Pathogenicity was assessed using SIFT (http://sift.jcvi.org/), PolyPhen-2 , LRT (http://www.genetics.wustl.edu/jflab/lrt_query.html), and MutationTaster (http://www.mutationtaster.org).Among the 20 novel cases found with AIFM1, while the variations were absent in our sensorineural hearing loss group (PS4). Secondly, these variations were absent or at extremely low frequency in ESP, ExAC, gnomAD-EAS, or 1000genomes (PM2). Thirdly, in these families, the variations were cosegregated with AN phenotype, with proband's mother carrying variation but having no AN performance (PM). Fourthly, these variants were predicted to be deleterious with SIFT, PolyPhen-2, LRT, MutationTaster, and so on (PP3). In addition, in terms of phenotype, these patients were all reported as AN(PP4). Furthermore, cells with the AIFM1 mutation led to decreased dimerization and impaired mitochondrial functions (unpublished data), which may indicate that the mutations in AIFM1 gene may affect auditory function, providing PS3 evidence.The seven novel variations were all pathogenic with the evidences of “PS×1 + PM×2 + PP×2” according to the ACMG and AMP guidelines . FirstlyThe recurrent variants were p.Ile304Met, p.Leu344Phe, p.Arg422Trp, and p.Tyr560His, among which the most common variant in the AN population was p.Leu344Phe, which was present in 36.1% (13/36) of the positive cases, followed by p.Arg422Trp .AIFM1 variations were related to the AN phenotype, with 9 variations located in FAD, 6 variations in NADH, and 4 variations in the C-terminal region. There was no overlap with the other AIFM1 variations that caused other syndromes [In total, 18 variations in yndromes –28  not complaining of childhood-onset AN. Except for one female case with unilateral AN, all other patients showed bilateral AN. The audiograms varied, with 31 ears  showing upsloping types. Seven of 20 patients underwent inclined sagittal MRI of the internal auditory canals, with 3 patients showing bilateral cochlear nerve hypoplasia.Except for families 0804755 and 1507328, who had a family history, 18 other cases with AIFM1 variations. Except for 0804755 , which was also the most common variant among AN-related  0804755 , the oth 0804755 . However 0804755 .Sixteen patients (53.33%) underwent follow-up pure tone audiometry tests, and the follow-up period was 1-23 years, with a mean time of 9.75 ± 9.89 years. The hearing threshold change varied between patients . The avep = 0.0096). This result suggested that the hearing threshold of 0.5 kHz in both ears worsened gradually over time. The hearing thresholds of 0.25, 1, 2, 4, and 8 kHz changed in a similar pattern as that of 0.5 kHz, although statistical significance was not reached in either ear  were followed up. A total of 10 patients (20 ears) were followed up for SDS (Table p < 0.05). Therefore, the degree of SDS decline in these patients with AN was not proportional to the pure tone hearing threshold, and SDS was more severe in patients with mild hearing loss than in patients in the moderate and severe hearing loss groups (Table p < 0.05). The mean values of the left and right ears before and after follow-up were greater than zero, indicating that the SDS exhibited a downward trend before and after follow-up, but the difference between the two ears was not statistically significant (p > 0.05).Twenty-three patients  with DS Table , with a ps Table . Diseaseps Table . This fip > 0.05)  Table . Three o5) Table .All 30 patients underwent DPOAE; 29 patients passed with the elicitation of at least five frequencies, while one patient had no response at any of the frequencies. This individual was a member of family 1007170 and had a disease duration of 33 years. AN may progress to sensorineural hearing loss, with outer hair cell impairment as time goes on.p < 0.001). For the 6 cases (12 ears) with a follow-up ECochG test  underwent electrocochleography examination, among whom, 6 patients underwent follow-up observation. The -SP waves were found in 21 patients, except for one person who had an unobvious wave. Nine patients  showed the -SP wave only, without an obvious CAP wave, while the remaining 35 ears showed both -SP and CAP waveforms, with absolute values of ‐SP/AP > 0.4 Table . The degt Figure , there wAN is a special type of hearing dysfunction disease, which is one of the critical diseases that cause speech communication disorders in infants and adolescents . In the OTOF is the most common gene-causing congenital auditory neuropathy [AIFM1 is reported to be the most common genetic cause [AIFM1 is the most common genetic cause of all noninfant-onset AN cases. The identification of genes is helpful to identify related lesion sites of AN and contributes to a better understanding of the underlying pathogenic mechanisms [As a difficult and popular topic in international research, research on AN has been performed for 20 years, from preliminary reports to various explorations of its pathogenesis, and it is beginning to be gradually understood accurately. The etiology of AN varies with age, genetic factors, hyperbilirubinemia, low birth weight, premature birth, and hypoxia. More patients may be discovered as the use of genetic testing in the diagnosis of auditory neuropathy becomes more widespread –8; howevuropathy . In contic cause . In thischanisms .The AIFM1 gene, also known as AIF, PDCD8, COXPD6, etc., is located in the human chromosome Xq25-q26 region, with a full length of 36.471 kb and 16 exons encoding a full-length 613 amino acid protein. In the mitochondria, AIFM1 acts as a FAD-dependent NADH oxidoreductase and plays a critical physiological role in the stable and mature mitochondrial oxidative respiratory chain complex I and the elimination of peroxide. In this study, we further expanded the mutation spectrum and long-term phenotypes of AIFM1-related cases. We found another 20 AN cases with AIFM1 variants by whole genome sequencing and Sanger sequencing, including 7 novel variants. All 18 AN-related variations had no overlap with the other phenotype-related AIFM1 variations (Table AIFM1 c.1030C>T (p.Leu344Phe). Due to its location in the loop region, this variation may have an influence on folding.ns Table . In addiAIFM1 variants, the phenotype of whom was similar to those of the male cases with the same variant. AIFM1 gene mutation AN may also be inherited in an X-linked dominant inheritance pattern. In our previous study, except for the patients undergoing whole exome sequencing, we did not pay attention to female AN cases; only male cases were tested for the AIFM1 gene.In this study, we found 5 female AN cases with AIFM1-related AN, a follow-up study was performed. Further genotype-phenotype correlation analysis can effectively help physicians and patients understand the disease-causing mechanism, process, and outcome of disease by research methods. To further assist in the consultation and evaluation of prognosis in patients with clinical AN, the clinical phenotypes of AIFM1-related cases were as follows:AIFM1-positive cases, that the low frequency spectra were mostly affected, especially in the 0.25-1 kHz range. Hearing impairment ranged from mild to moderate. No significant differences were detected in the hearing thresholds tested between the first and final visits within 1-10 years of follow-up. However, when the follow-up periods were prolonged to over ten years, hearing thresholds in both the low frequencies (0.25-0.5 kHz) and the high frequencies (4-8 kHz) showed significant worsening. In addition, the pure tone threshold tended to deteriorate over time, especially at a frequency of 500 HzFor the pure tone threshold, AN patients have large individual differences in audiologic phenotype. Although the ascending audiogram is the typical audiometric pattern of AN patients, audiograms with various configurations and varying severities may occur. The hearing of some patients may improve, while others' hearing loss may remain stable for a long time or even worsen. Among the very limited reports involving the follow-up characteristics of AN patients, the hearing outcomes in a long-term follow-up remain elusive . Not surAIMF1 gene-related AN is gradually aggravatedFor SDS, the AN patient's prominent complaint is that he/she can hear voices without understanding the meaning. Our study found that patients with mild hearing loss had a high proportion of a score of zero when testing SDS, which was much more severe than the SDS in the moderate and severe hearing loss groups, and the degree of SDS was not consistent with the pure tone threshold. This fact suggests that in clinical audiology assessment, the SDS performance of AN patients is much more critical than that of PTA. The evaluation of the degree of hearing loss by PTA alone may underestimate the patient's condition. Furthermore, we followed up on the SDS of this type of AN patient and found that SDS decreased significantly with the prolongation of the disease course. This result indicates that the neurological synchronization of the AIFM1 gene mutation may be located in the auditory conduction pathway outside the inner hair cells. CAP is produced in cochlear spiral ganglion cells and is an afferent nerve response. The decrease in CAP amplitude can prove the synchrony decline in auditory nerve activity. We observed that the -SP and CAP waveforms were present in 80% of patients at the same time, but the absolute value of -SP/AP was >0.4, which was higher than normal, suggesting that there was a loss of synchronization of auditory nerve activity. This also explains why the SDS decline in this type of patient is more marked than the decrease in PTA from the perspective of physiology and pathology. Furthermore, we analyzed the patient's auditory condition based on the presence or absence of CAP waves, showing that the degree of hearing loss without CAP waveform was more serious. Clinically, the detection value should be increased, and the degree of hearing loss should be comprehensively judgedECochG revealed that -SP and CAP waves existed together in 80% of cases, but the SP/AP values were higher than normal. Patients showed worse hearing loss when their CAP waves disappeared. Compared with DPOAE, cochlear electrograms can help us locate the lesions of AN . The -SPTo decipher the phenotype progression of AIFM1-related AN suggested that auditory dyssynchrony progressively worsened over time. Electrophysiological examinations of the cochlear nerve indicated that lesions would be located on the auditory pathway from postsynapses to acoustic fibers [AIFM1 mutation may have poor efficiency from cochlear implantation.Individuals with lesions affecting the auditory nerve showed poor performance with cochlear implantation, since the neural transmission was affected . The audc fibers . Furtherc fibers , 46; froAIFM1 mutation-related hearing loss is still unclear; the mitochondrial function and the caspase-independent apoptosis to neuronal development and adult neurogenesis play a critical role in previous studies [AIFM1 mutation led to decreased dimerization and further impaired mitochondrial functions, such as increase of ROS production and impairment of mitochondrial membrane potential, thereby activating caspase-independent apoptosis (unpublished data). However, the complete and clear pathogenesis of AN and the genotype-phenotype correlation need to be further clarified.The molecular disease-causing mechanism of  studies , 48. In AIFM1 is the most common genetic cause of late-onset AN, with the hotspot mutation of c.1030C>T (p.Leu344Phe). (2) In addition to the X-linked recessive inheritance pattern, the X-linked dominant inheritance pattern is another probability of AIFM1-related AN, which affects females. (3) The hearing threshold of AN patients with AIFM1 mutation tends to worsen when the follow-up period is prolonged. Phenotype features of AIFM1-related AN suggested that auditory dyssynchrony progressively worsened over time. Electrophysiological examinations of the cochlear nerve indicated that lesions would be located on the auditory pathway from postsynapses to acoustic fibers.In conclusion, (1)


Objective: To design a multi-targeted fecal DNA methylation kit and explore its value for clinical application among Chinese people.Methods: Based on previous research, a multi-targeted fecal DNA methylation detection kit, using four genes, was designed and clinically validated.Results: The methylation PCR from 279 patients met the requirements for the detection criteria. When all four molecular markers were negative, the negative predictive value (NPV) for colorectal cancer was 100% and the NPV for colorectal polyps was 84.21%.When one molecular marker was positive, the sensitivity (Se) for colorectal cancer was 76.4%-90.3%, the specificity (Sp) was 68.3-93.4%, and the positive predictive value (PPV) for colorectal cancer was 54.5-85.5%, and the NPV was 87.0-95.0%. For colorectal polyps, the Se was 41.0-52.5%, Sp 69.5-91.5%, and the PPV for colorectal polyps was 41.0-70.3%, the NPV was 75.2-79.3%.When two molecular markers were positive, the Se for colorectal cancer was 52.6-73.7%, the Sp was 93.2-98.3%, the PPV for colorectal cancer was 84.6-96.2%, the NPV was 76.0-85.3%. For colorectal polyps, the Se was 25.9-40.7%, Sp was 93.2-98.3%, PPV for screening of colorectal polyps was 63.6-90.0%, and the NPV was 73.3-78.1%.When three molecular markers were positive, the Se for colorectal cancer was 31.6-52.6%, the Sp was 98.3-100.0%, the PPV for colorectal cancer was 94.4-100.0%, the NPV was 73.4-76.6%. For colorectal polyps, the Se was 14.8-25.9%, and Sp was 98.3-100.0%, the PPV for colorectal polyps was 85.7-100.0%, the NPV was 72.0-74.7%.When four molecular markers were positive, the Se for colorectal cancer was 31.6%, the Sp was 100.0%, and the colorectal cancer PPV was 100.0% and the NPV was 69.4%. For polyps, the Se was 14.8%, Sp was 100.0%, and PPV was 100.0% and the NPV was 72.0%.Conclusion: The multi-targeted fecal DNA methylation detection kit for colorectal cancer and polyps had the sensitivity and specificity to meet the requirements for screening of colorectal tumors, which is easy to operate, has stable results and important clinical value. Among the four molecular markers studied, when one marker was positive for DNA methylation, colonoscopy was required; as the number of positive methylation markers increased, the specificity for the diagnosis gradually increased as well. Colorectal cancer is preventable and curable. However, the incidence and mortality associated with colorectal cancer has not decreased significantly. Tumor stage is still an independent risk factor associated with colorectal cancer. The most effective way to prevent colorectal cancer and improve clinical outcomes is the detection of colorectal polyps and early stage colorectal cancer In theory, FOBT can only be detected by bleeding in the intestine. Although iFOBT can detect bleeding as low as 100ng/ml The gut mucosa is regularly renewed; cells from this mucosa are shed with stool. Screening for colorectal tumors, using DNA molecular markers on the mucosa that is present with stool, might be an effective way to screen for colorectal cancer. Imperiale et al. In this study, SNCA, SPG20, Septin9, and FBN1 were selected as molecular targets PCR primers were designed for the regions of Septin9, SNCA, SPG20, and FBN1 without CpG double bases as in our previous reports 279 cases were enrolled in this study. Sixty-two patients with colon cancer, 71 cases with colon adenoma and 146 patients with a normal colonoscopy at the Second Affiliated Hospital of Nanjing University of Traditional Chinese Medicine were recruited into the study from May 2016 to May 2020. All patients signed the informed consent, and the study obtained Approval by the Ethics Committee of the Second Affiliated Hospital of Nanjing University of Traditional Chinese Medicine (KY2014018). Participants' clinical data are shown in Table All stool samples were handled in a blinded fashion during storage and processing. DNA extraction and qMSP analysis were performed. Patient samples were collected and processed according to the method previously published by our group (1) Evaluation of experiment quality: If there was no amplification or Ct value in the detection channel of the negative control, the analysis was continued; otherwise, the experiment was considered invalid and had to be repeated.(2) Evaluation of the positive and negative control gene detection: If the positive control channel had amplification and the Ct value was ≤ 25, the analysis was continued. If the positive control channel had a large Ct value or no amplification, but the mutation site was detected and amplified and the Ct value was ≤ 38, the analysis was continued. If the positive control channel had a large Ct value or no amplification, and the mutation site was detected without amplification or there was amplification but the Ct value was > 38, the analysis could not be continued; this indicated that the experiment had failed, and needed to be repeated. If the negative control gene control channel showed amplification, this implied that the experiment had false positive results and had to be repeated.(3) Judgment of gene mutation in the DNA sample to be tested: if the detection channel of a mutation site in the sample was amplified and the Ct value was ≤ 35, the mutation result of the sample was considered positive. If the Ct value was > 38, or no amplification was noted, then the mutation result of the sample was determined to be negative. If 35 < Ct value ≤ 38, the experiment needed to be repeated. If the Ct value was still within this range, the sample mutation result was suspected to be positive  Figure .The data was processed with SPSS 18.0. The count data was tested by the Chi-square test or Fisher's exact test. The measurement data was expressed by mean ± SD and the t-test was used. Receiver operating characteristic (ROC) analysis was used for the marker gene diagnosis Value; The sensitivity, specificity, positive predictive value, and negative predictive value of each index was calculated using the results of colonoscopy as the “gold standard”.Detection of methylation in all 279 patients met the criteria requirements and the results could be analyzed. The methylation rates of SNCA in colorectal cancer, polyps and normal mucosa were 90.32%, 46.47% and 14.38% respectively. The methylation rates of SPG20 were 85.48%, 56.33% and 27.40% respectively. The methylation rates of FBN1 were 79.46%, 50.70% and 13.01% respectively. The methylation rates of Septin9 were 91.93%, 56.33% and 6.16% respectively. The stages were divided into groups I-II, III-IV and polyps' sizes were <0.5cm, 0.5-1cm and ≥1cm. The polyps were divided into tubular, tubulovillous and villous according to the pathological features. The p-values of the methylation among different targets in colorectal cancer, polyps and normal mucosa were included in Table Using the colonoscopy results as the gold standard, we calculated Se, Sp, PPV and NPV of one, two, three, or four molecular markers with methylation positive for colorectal cancer, colorectal adenomas, and colorectal cancer and adenomas. When all four molecular markers were negative, the NPV for colorectal cancer was 100%, and the NPV for colorectal adenomas was 84.21%.When one molecular marker was positive for DNA methylation, the methylation rate for four molecular markers, in colorectal cancer and adenoma, was higher than that of patients without these findings, and the difference was statistically significant. The Se, Sp, PPV and NPV of one molecular marker positive for colorectal cancer and adenoma screening are shown in Table When two molecular markers were positive for DNA methylation, the difference between cancer and adenomas was significantly different from normal mucosa. The specificity of screening for cancer or polyps was significantly increased, reaching more than 93%; however, in such cases the sensitivity decreases. The Se of cancer screening was 52.6% -73.7%, and the Se of adenoma screening was 25.9-40.7%, with PPV increasing and NPV decreasing. Table When 3 or 4 molecular markers were positive for DNA methylation, the differences between cancer and adenoma was significant when compared to normal mucosa. The specificity of screening for cancer or adenomas was close to 100%, but the sensitivity decreased. The sensitivity for cancer screening was 31.6-52.6%, and the sensitivity for adenoma screening was 14.8-25.9%. The PPV increases and the NPV decreases. Table iFOBT had a higher sensitivity and specificity in colorectal cancer screening and plays an important roles in detection of early stage colorectal cancer, theoretically, iFOBT can only find the blood in the intestinal cavity. Any diseases with intestinal bleeding such as enteritis, diverticulitis, inflammation bowel disease (IBD) and so on, may produce false positive; at the same time, if the colon neoplasm does not bleed, it will appear false negative. Table The detection of tumor molecular markers in stool is a valuable screening method for colorectal cancer. It is also easily accepted by patients because of its non-invasiveness and low cost We designed a multi-targeted DNA methylation screening kit for colorectal tumors. We used positive and negative methylation controls to perform quality control on the reaction system, thereby eliminating false positives and false negatives and increasing the accuracy of screening. In this study, the test results of all 279 patients, the positive controls and the negative controls were in line with the design, suggesting that the results of this group of studies were reliable.For the specificity of detection, we used four targets for detection at the same time. When all four molecular marker DNA methylation tests were positive, whether it was an adenoma, cancer or cancer plus adenoma, the specificity of diagnosis was 100%, and the positive predictive value is 100%. In other words, if all four signs were positive, the patient was thought to have colorectal cancer and/or adenoma; colonoscopy and treatment followed. Using the Cologuard kit, the sensitivity for colorectal cancer was 92.3%, and the sensitivity for colorectal polyps was 42.4%; the specificity was 86.6% In our study, when three out of four markers were positive, the specificity was more than 98.3%, and the positive predictive value was more than 85.7%; which means that when performing colonoscopy, a "negative" colonoscopy would be predicted in 15% of cases or less. The efficiency of colonoscopy was significantly improved. When two molecular markers were positive, the specificity was more than 93.2%. Except that the PPV for SNCA + SPG20 and SNCA + FBN1 were slightly lower, the PPVs of other combinations reached more than 80%. If a colonoscopy was performed, the positive rate was high. When one markers was positive, the specificity for colorectal cancer ranged from 68% to 93%, the specificity for colorectal adenoma was ranged from 77% to 99%. Especially when the FBN1 and SEPT9 were positive, the specificity were higher. The PPV of a colorectal tumor (cancer+adenoma) was more than 71%. If a colonoscopy was performed in these patients, only 30% of the examinations were "negative". SPG20 was with lower specificity, we will optimize the molecular markers according to the study.From a sensitivity perspective, the more markers applied, the lower the sensitivity. For example, when all four markers were positive, the sensitivity for colorectal cancer was only 31.6%, and the sensitivity for colorectal polyps was only 14.8%. When two or three markers were positive for DNA methylation, the sensitivity for colorectal cancer and adenoma was only 24.6%. However, when one molecular marker was used for DNA methylation, the sensitivity increased. When a single molecular marker was positive, the sensitivity for colorectal cancer was 76.4% to 90.3%, the sensitivity for colorectal adenoma was 41.0% to 52.5%, and the sensitivity of colorectal cancer and adenoma was 72.6% to 99.2%, which is ideal for molecular markers. Therefore, in terms of sensitivity, as long as one of the four markers was positive and the positive predictive value of colorectal cancer and adenoma was more than 71%, the recommendation was for the patient to have a colonoscopy.When all four molecular markers were negative, the NPV for colorectal cancer was 100%, and the NPV for colorectal polyps was 82.8%. That is, when all four molecular markers were negative, the test subject had a risk for colorectal cancer of zero, and the risk of colorectal polyps was 17.2%. They don't need a colonoscopy for colorectal cancer screening.In summary, this study confirmed that a multi-targeted fecal DNA methylation screening kit has the sensitivity and specificity to meet the requirements of screening for colorectal cancer. It is easy to perform, has stable results, and important clinical value. When one of four markers was positive for DNA methylation, colonoscopy was recommended. As the number of methylation markers increased, the specificity of diagnosis gradually increased. When more than two markers were positive for DNA methylation, the patient's PPV was over 90%, which indicated a very high risk of colorectal cancer or adenoma. Colonoscopy and treatment were needed immediately to detect colorectal cancer or adenoma. When all four molecular markers were negative, the possibility of colorectal cancer was very low.


Hearing loss is one of the most common sensory deficits. It carries severe medical and social consequences, and therefore, universal newborn hearing screening was introduced at the beginning of this century. Affected patients can have hearing loss as a solitary deficit (non-syndromic hearing loss) or have other organs affected as well (syndromic hearing loss). In around 60% of cases, congenital hearing loss has a genetic etiology, where disease-causing variants can change any component of the hearing pathway. Genetic testing is usually performed by sequencing. Sanger sequencing enables analysis of the limited number of genes strictly preselected according to the clinical presentation and the prevalence among the hearing loss patients. In contrast, next-generation sequencing allows broad analysis of the numerous genes related to hearing loss, exome, or the whole genome. Identification of the genetic etiology is possible, and it makes the foundation for the genetic counselling in the family. Furthermore, it enables the identification of the comorbidities that may need a referral for specialty care, allows early treatment, helps with identification of candidates for cochlear implant, appropriate aversive/protective management, and is the foundation for the development of novel therapeutic options. Hearing loss is the most common sensory deficit, as 1 in 1000 neonates have severe to profound, permanent hearing loss .We can divide hearing loss by the place where it occurs. When it is caused by the malfunctioning of the outer and middle ear (auricle to the cochlea), it is addressed as conductive hearing loss (CHL). On the other hand, when the reason is in the cochlea, auditory nerve, auditory nuclei, or auditory cortex, we use the term sensorineural hearing loss (SNHL). In the mixed hearing loss, both causes are present at the same time.Regarding the timing of the onset, hearing loss can occur before the acquisition of speech—prelingual, after the acquisition of speech-post lingual, in adulthood- adult-onset or later; age-related onset or presbycusis. Regarding progression, hearing loss can be progressive, nonprogressive, or fluctuating. It can be unilateral or bilateral. The pure tone audiogram curve could have a sloping, flat, rising, or mid-frequency loss (cookie bite) configuration, which is essential in genetic diagnostics of hearing loss . While tUnilateral and bilateral hearing loss have severe consequences on a child’s development if left untreated. They might negatively affect language development, classroom learning, academic performance, and social development . UnilateThe fact that the majority of newborns have a hereditary cause for their hearing loss is being obscured by the fact that ninety-five percent of newborns with hearing loss identified by newborn hearing screening programs are born to hearing parents .The majority of congenital hearing loss, up to 60% of cases, is due to a genetic etiology . HearingNumerous genes are related with non-syndromic hearing loss that can be transmitted in an autosomal recessive, autosomal dominant, x-linked, or mitochondrial pattern of inheritance .Autosomal-recessive (AR) inheritance accounts for 80% of non-syndromic genetic hearing loss, predominantly being prelingual . AutosomGJB2, which encodes the protein connexin 26 [GJB2 deficiency was first described in 1997, and since then, worldwide GJB2 coding region sequencing has demonstrated various spectrums of genotypes across different populations. A particularly prevalent GJB2 disease-causing variant is a deletion NM_004004.6: c.35del (p.Gly12fs) with a carrier frequency 1/35 in southern and 1/79 in central and northern Europe [GJB2 gene are present in 26.6% of patients with congenital hearing loss [GJB6 gene are the second most common reason for prelingual SNHL in some populations (like Spanish) [In the case of AR inheritance, there are usually no other family members with the same type of hearing loss. Most of the patients with non-syndromic SNHL have the disease-causing variant in the gene nexin 26 , a gap jnexin 26 . Hearingn Europe  and 1/72n Europe . In the ing loss  and 22.2ing loss . While lSpanish) , no suchSpanish) .TMPRSS3 gene, encoding a transmembrane protein that belongs to the serine protease family, are the cause for post lingual or congenital hearing loss [GJB2 or GJB6 genes had disease-causing variants in the TMPRSS3 gene [Causative variants in the ing loss . As muchSS3 gene .TECTA gene is linked to AD non-syndromic hearing loss and disease-causing variants in the TECTA gene are the most frequent cause of dominant hearing loss [The ing loss . It encoing loss . It is aing loss .Hearing disorders with mitochondrial inheritance patterns frequently result in a syndromic presentation. The exceptions are causative variants in the mitochondrial 12s ribosomal RNA (MTRNR1)  associatPOU3F4 variants. A defective POU3F4 gene is clinically characterized by cochlear hypoplasia, bulbous internal auditory canals, and sensorineural hearing loss with a variable conductive component [X-linked non-syndromic hearing loss, however, is commonly caused by omponent .MYO7A, associated with the abovementioned Usher syndrome and rarely to hearing loss, with various severity and autosomal recessive (DFNB2) or autosomal dominant (DFNA11) inheritance [In some cases, different disease-causing variants in the same gene can be associated with hearing loss inherited in recessive or dominant mode. An example of such a gene is eritance . The clieritance .Hearing loss is among the most etiologically heterogeneous disorders, with more than 400 genetic syndromes that include hearing loss as a feature . SyndromSLC26A4 gene  may be very important for the child’s future. It may help to predict whether the child will develop thyroid dysfunction or goiter after puberty, or might be at risk for hearing loss after head trauma due to an enlarged vestibular aqueduct [In patients with suspected syndromic hearing loss, it is often even more important to determine the genetic cause, as many of the accompanying clinical features can be more severe. Early diagnosis can predict the progression of hearing loss, guide future treatments, and provide a warning for potentially life-threatening abnormalities . For insaqueduct . Some paaqueduct .USH2A gene [MYO7A gene encodes myosin 7A and is causative for 75% of Usher syndrome type I cases [USH2A gene were reported, as much as 84% of the Slovenian patients with Usher syndrome type 2 had the NM_206933.4:c.11864G>A (p.Trp3955Ter) variant [The most common among autosomal recessive syndromic hearing losses is Usher syndrome with the prevalence 1/6000 to 1/25,000 . It is aH2A gene , encodin I cases . A large I cases . Althoug variant . As blin variant , as it yJervell and Lange-Nielsen syndrome is the third most common cause of autosomal recessive syndromic hearing loss due to variants in genes encoding for voltage-gated potassium channels ,46. The NF2) gene encoding the tumor suppression protein [EYA1 and SIX5 genes [Among an autosomal dominant group of syndromic SNHL, the most common is neurofibromatosis type 2 (NF2) with bilateral vestibular schwannomas, meningiomas, and optical gliomas . The inc protein . Another protein . Mild toX5 genes . Other wX5 genes .GJB2, commonly mutated in NSHL patients or SLC26A4, associated with a particular clinical feature, are still often analyzed with this approach. On the other hand, next-generation sequencing (NGS) enables better sequencing performance with the simultaneous reading of numerous sequences [GJB2 subgroup [GJB2 has been pre-screened, the diagnostic rate increases to approximately 50% [The conventional sequencing method in genetic diagnostics of any inherited trait is the Sanger dideoxy or enzymatic chain termination method . This apequences . With thequences . Alternaequences . NGS seqequences . This misubgroup . With thtely 50% .The result of sequencing is a nucleotide sequence that needs to be compared to the reference sequence of the analyzed gene, such as those collected in the NCBI Genome database , which iRegarding the known data about the identified sequence variant, the decision regarding its impact on the patient’s clinical picture should be made. Each variant should be categorized as a benign, probably benign, variant of unknown significance, probably pathological, or pathological . NeverthIn the University Medical Centre Ljubljana, newborns with suspected hearing loss identified by the UNHS program are referred to the Otorhinolaryngology Clinic—Audiovestibulology department outpatient consultation. History taking and a detailed clinical otoneurologic exam (including otomicroscopy) are performed, followed by further hearing testing. As a first step, tympanometry, which helps to define the outer and middle ear function, is performed. Due to the unresponsiveness of TOAE even in mild hearing loss cases, we proceed with TOAE testing only in cases with a good tympanometry result—namely tympanogram type A or eventually C. In cases with no response to TOAE tests, we proceed with audiometry in the free field  using warbled pure tones sounds across the frequencies of 250 to 4000 Hz. Specially trained clinical speech and language therapists perform these tests. In the case of a child’s poor responsiveness, we consider the introduction of a hearing aid. All children with tympanogram A and non-evoked TOAE are objectively tested in induced sleep .GJB2 and GJB6 coding regions followed by targeted NGS sequencing of syndromic and non-syndromic hearing loss-related genes. See the genetic hearing loss algorithm in At the audiovestibulology department of the University Medical Centre Ljubljana, the auditory steady-state response (ASSR) is measured. As a result, we obtain an audiogram-like curve that helps us evaluate the degree of the child’s hearing loss. Some children are referred to the Children’s Hospital neurophysiology laboratory for the measurement of auditory brainstem responses. If permanent hearing loss is identified, an effort is made to provide the child (with the parent’s consent) with a hearing aid by the age of four to six months. If a child is not making progress or has been diagnosed with severe-to-complete hearing loss, cochlear implantation by the age of 11–12 months is planned. All children with suspected or proven hearing loss or children with factors that endanger them for the possible development of hearing impairment have a regular follow up at the Audiolovestibulology centre, where we measure their hearing and speech and language progress and appropriately adjust our interventions. The protocol of patient workup is summarized in Hearing is essential for linguistic, social, and intellectual development. Consequently, hearing restoration is a crucial role of audiologists and ENT specialists. In most hard of hearing patients, auditory rehabilitation includes conventional hearing aids. Cochlear implantation (CI) is the surgical insertion of an electrode that provides electrical stimulation directly to the auditory nerve (bypassing the cochlea in which the pathogenetic cause lies). At the present time, CI is the standard therapeutic option for severe-to-profound sensorineural hearing loss patients . From thInspired by advances in knowledge regarding the genetic etiology of SNHL and the successes in other specialties, there is a growing interest in gene therapy for hearing loss . It is bThe translation from animal models into gene therapies for humans with genetic deafness has not yet begun. Nevertheless, in other systems, there are numerous (1800) clinical trials that have been either completed, are in progress, or approved regarding gene therapy . AnotherThere are still numerous unanswered questions regarding gene therapy for hearing loss in humans. Still, despite that, the field of inner ear gene therapy is moving toward the goal of developing a powerful therapeutic tool for patients with genetic hearing loss .When a genetic basis of hearing loss is established, genetic counselling as part of the extended clinical genetics evaluation is offered to patients and their families . There aAlthough CI provides a beneficial outcome for most of the hearing loss/deafness cases, factors affecting the results of CI vary among patients. The heterogeneous cause of hearing loss is thought to be one of the reasons for such variations . Good CIPatients with specific hearing loss-related syndromes develop other comorbidities later during childhood or life. It is crucial to make the etiological diagnosis early to enable regular monitoring of the function of other organs . That woFurthermore, genetic testing enables other clinical interventions. The examples are early treatment such as dietary supplementation with S-adenosylmethionine in PRPS1 deficiency and treatment for hypocalcemia in hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome . Furtherde novo variants, inherited variants carry a high recurrence risk and that enables options for prenatal diagnosis. It is also vital to understand the limits of genetic testing. When the result of extensive genetic testing is negative, we have to keep in mind that hearing loss could still have genetic etiology, since the disease-causing variant may be located in the region that has not been tested [Genetic counselling helps family members in dealing and accepting the diagnosis and its consequences. It is crucial in explaining the genetic risk for relatives and future offspring . Unlike n tested .Hearing loss is one of the commonest sensory deficits. In around 60% of cases, congenital hearing loss has genetic etiology. The progress of medicine has made early genetically diagnostic of children with hearing loss possible. It is essential to make an early etiological diagnosis of hearing loss as it can have numerous significant consequences on the affected child’s and its family’s health and quality of life. Nevertheless, in numerous cases, the prediction of the phenotypic outcome based on the genetic background is difficult and imprecise and therefore, merits additional studies.


Absent and/or prolonged VEPs were observed in 10/13 (76.9%) patients and 17/26 (65.4%) eyes. On MRI, hypertrophy, and high signal intensity of trigeminal nerves were detected in 9/13 (69.2%) and 10/13 (76.9%) patients, respectively, whereas optic nerves were normal in all patients. The intra‐orbital trigeminal nerve width on coronal sections showed a significant positive correlation with disease duration.Among 13 patients, facial sensory impairment, facial weakness, and apparent visual impairment were observed in three (23.1%), two (15.4%), and two (15.4%) patients, respectively. All 12 patients tested had blink reflex abnormalities: absent and/or delayed R1 in 11 (91.7%), and absent and/or delayed R2 in 10 (83.3%). R1 latencies had strong positive correlations with serum anti‐NF155 antibody levels (+ CIDP, suggesting that both central and peripheral myelin structures of the cranial nerves are involved in this condition, whereas nerve hypertrophy only develops in myelinated peripheral nerve fibers.Subclinical demyelination frequently occurs in the optic, trigeminal, and facial nerves in IgG4 NF155 Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired immune‐mediated disease of the peripheral nervous system (PNS) and constitutes the most prevalent chronic autoimmune neuropathy.+) CIDP.Accumulating evidence indicates that some patients with CIDP harbor autoantibodies against nodal or paranodal proteins, such as neurofascin 186 (NF186), neurofascin 155 (NF155), contactin 1 (CNTN1), and contactin‐associated protein 1.+ CIDP were reported, whereas others did not detect anti‐NF155 antibodies in CCPD.+ CIDP is unclear and the differential effects of anti‐NF155 antibodies on peripheral myelin/Schwann cells and central myelin/oligodendrocytes remain to be established. Therefore, in this study, we aimed to characterize cranial nerve involvement in IgG4 NF155+ CIDP by neuroimaging and electrophysiology, with the trigeminal and facial nerves representing peripheral myelin structures and the optic nerve representing a central myelin structure.Although overt central nervous system (CNS) manifestations are rare in CIDP, CNS tissues including cerebral white matter and optic nerves could be involved. Various names have been used to describe such conditions, for example, combined central and peripheral demyelination (CCPD).+ CIDP diagnosed and treated in our department since 2001, 13 patients who underwent head magnetic resonance imaging (MRI) and blink reflex or visual‐evoked potential (VEP) testing were enrolled. All patients met the European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) diagnostic criteria for CIDP.Of 15 consecutive patients with IgG4 NF155IgG and IgG4 anti‐NF155 antibodies, and IgG anti‐NF186, and anti‐CNTN1 antibodies in sera, were measured by flow cytometry using human embryonic kidney 293 cell lines stably expressing human NF155, NF186, or CNTN1 as described previously.2 with a contrast level of 90%. Subjects were instructed to fixate their eyes at the center of the stimulus field at a viewing distance of 100 cm. Monocular full visual fields were stimulated. VEPs were recorded from an electrode placed at Oz with a reference at Fz . A total of 100 responses were averaged and recordings were repeated at least twice to establish reproducibility. The latencies of the major positive peak in each check size were measured (P100 15’ and P100 30’). The upper normal limits  of VEPs were as follows; P100 15’, 123.8 ms; P100 30’, 121.0 ms.Nerve conduction studies (NCSs) were performed with standard surface stimulation and recording techniques . Motor nerve conduction studies, including F wave analyses, were investigated in unilateral median and ulnar nerves. Skin temperature was maintained above 31°C in the upper limbs. Base‐to‐peak amplitudes were measured for CMAPs. Blink reflex was recorded with stimulation at the supra‐orbital notch and recording over the orbicularis oculi muscle on the inferior rim of the orbit directly below the pupil. Recordings were superimposed, and the shortest R1 and bilateral R2 latencies were used for each patient.Abnormalities of size and signal intensity in the first V1) and second (V2) branches of trigeminal nerves in the orbit and the third branch (V3) at the level of the foramen ovale were evaluated by axial and/or coronal views of T2‐weighted images (T2WIs), including T2 spectral adiabatic inversion recovery images. Axial images were taken in all patients, whereas coronal images were taken in eight patients . Welch’s t‐test was used to compare the time from disease onset to MRI between patients with and without trigeminal nerve hypertrophy. The correlations between blink reflex and NCS results and other clinical parameters and between the maximum width of the intra‐orbital trigeminal nerve on each side and periods from disease onset to MRI were assessed using Pearson’s correlation coefficients. and in 21 of 24 sides (87.5%) tested  and 17/26 eyes (65.4%) to have abnormalities  had abnormal findings of the trigeminal nerves on axial and/or coronal T2WIs  had an abnormal blink reflex but normal VEPs and one patient (Case 10) had a normal blink reflex but abnormal VEPs. Parameters of the blink reflex did not correlate with age at onset, age at blink reflex test or the time from disease onset to the blink reflex test.r = 0.7713, P = 0.0149; left: r = 0.8834, P = 0.0036) and F wave latencies , and negatively correlated with motor conduction velocities , and tended to positively correlate with DLs  and to negatively correlate with MCVs . Neither iR2 nor cR2 latencies showed correlation with CMAP amplitudes. Similar trends were observed with left side stimulation, albeit they were not significant. Similar correlations were also found between the blink reflex and median nerve NCS parameters (data not shown). Correlation analysis of the blink reflex parameters and serum anti‐NF155 antibody levels revealed that R1 latencies on stimulation of either side  and iR2 and cR2 latencies on right side stimulation  were positively correlated with MFI ratios . However, no correlation between VEP abnormalities and somatic NCS parameters was found.When the relationship between VEPs and other clinicolaboratory parameters was investigated, P100 15’ latencies of the right eye positively correlated with age at onset and age at VEP examination (age at onset: P = 0.1752). Additionally, the intra‐orbital trigeminal nerve width on coronal sections showed a significant positive correlation with disease duration  CIDP cases, particularly in the reports of Caucasians. Given that all NF155+ CIDP patients examined in this study had abnormal blink reflex, a higher frequency of blink reflex abnormalities is indicated for NF155+ compared with NF155‐ CIDP. The relatively higher frequency of blink reflex abnormalities in Japanese total CIDP patients+ CIDP in Asians+ CIDP.Concerning cranial nerve involvement in CIDP, blink reflex testing has repeatedly been shown to be helpful. Blink reflex test abnormalities in CIDP were reported to occur in 53.3% (8/15)Interestingly, our study showed a significant positive correlation of serum anti‐NF155 antibody levels with various blink reflex parameters, which suggests that high titer serum anti‐NF155 antibodies worsen conduction delay in the trigeminal and facial nerves more severely than low titer anti‐NF155 antibodies. This is consistent with the previous observation that anti‐NF155 antibody levels changed in parallel with MCV and F wave latencies in somatic nerves.A much higher frequency of R1 abnormality in the blink reflex test than trigeminal nerve hypertrophy indicates that subclinical demyelination precedes nerve hypertrophy.+ CIDP patients do not complain of visual disturbance, as described here and in previous reports.+ CIDP series is higher than the reported abnormal VEP frequencies in total CIDP patients,‐ CIDP cases, given the low frequency of NF155+ CIDP in Caucasians.The high frequency of VEP abnormalities is rather surprising, given that most NF155+ CCPD and CIDP patients.+ CIDP, which could be overlooked in clinical practice unless the blink reflex and VEPs are examined.For VEP analysis, the monocular full visual field was stimulated; therefore, marked prolongation of P100 latencies in the absence of ophthalmological retinal lesions and occipital MRI lesions suggests the presence of subclinical demyelination in the optic nerves in most of our cases. NF155 is expressed in the terminal loop of not only Schwan cells but also oligodendrocytes;+ CIDP, but that PNS and CNS involvement does not proceed in parallel. The observation that the correlation between R1 latency and NCS parameters seems to be stronger than that between R2 latencies and NCS parameters may be explained by longer CNS portions in the R2 compared with the R1 response.+ CIDP, DL‐ and F‐wave latencies are affected most severely,+ CIDP.The analysis of correlation between cranial and somatic NCS findings revealed that blink reflex parameters, particularly R1 latencies, were strongly correlated with NCS findings in the ulnar and median nerves, whereas no correlation existed between VEP and NCS findings. We therefore suggest that both the PNS and CNS are commonly afflicted in NF155+ CIDP, nerve hypertrophy frequently occurs in the trigeminal nerve branches but not in the optic nerves even in the same intra‐orbital cavity, although both nerves suffer from subclinical demyelination with similarly high frequency. The involvement of cranial nerve hypertrophy only in the PNS portion is also supported by our observation that the initial segments of the trigeminal nerve covered by oligodendrocytes did not show hypertrophy in any patient. Cranial nerve hypertrophy is sometimes visible on MRI in CIDP.+ CIDP,+ CIDP case.+ CIDP with a longstanding clinical course, which is helpful for suspecting this condition. Hypertrophy of cervical and/or lumbosacral nerve roots was seen in all 13 patients examined, whereas trigeminal nerve hypertrophy was detectable in around three‐quarters of the patients, which indicates that somatic nerve hypertrophy precedes cranial nerve hypertrophy. As in the somatic nerve,+ CIDP is suggested to gradually proceed over a long disease course in the cranial nerves belonging to the PNS but not the CNS, along with somatic nerve hypertrophy.Our study clearly shows that in NF155+ CIDP patients showed only perineurial and endoneurial edema without onion bulb formation or inflammatory cell infiltration,+ CIDP,Pathology of hypertrophic intra‐orbital nerves from some CIDP patients demonstrated onion bulb formation reflecting repeated demyelination and remyelination, infiltration of inflammatory cells, and abundant mucopolysaccharide deposition.+ CIDP. Thus, our study results should be confirmed by future large‐scale studies. Third, as we could not obtain sufficient data on blink reflex, VEP or brain MRI in NF155‐ CIDP patients, we compared between historical study results of total CIDP patients and NF155+ CIDP data. Thus, abnormality frequencies in these tests should be compared between NF155+ and NF155‐ CIDP patients in future research. Fourth, we did not have sufficient longitudinal blink reflex or VEP data for NF155+ CIDP. Thus, changes in subclinical demyelination in the optic, trigeminal, and facial nerves in response to immunotherapy should be longitudinally examined in the future.This study has several limitations. First, this is a retrospective study; therefore, slice thickness was not unified for the evaluation of cranial nerve hypertrophy and some clinicolaboratory data, such as corneal reflex and coronal T2WIs, were not available in several patients. Thus, clinical manifestations and nerve hypertrophy of cranial nerves could be under‐estimated. Second, the sample number was small because of the rarity of IgG4 NF155+ CIDP. Our study underscores the importance of awareness of the frequent involvement of the cranial nerves, including optic, trigeminal, and facial nerves, in NF155+ CIDP to prevent long‐term damage to these cranial nerves by immunotherapy.Nonetheless, we uncovered high‐frequency subclinical blink reflex and VEP abnormalities as well as trigeminal nerve hypertrophy, which suggest homogeneity of NF155Nothing to report.HO, XZ, SI, KY, RY, TM, NI, and JK designed and conceptualized the study, collected the data, analyzed the data, and drafted the manuscript. AH and ST analyzed the data and drafted the manuscript.


Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by a deficiency of one of the enzymes involved in the degradation of glycosaminoglycans. Hearing loss is a common clinical presentation in MPS. This paper reviews the literature on hearing loss for each of the seven recognized subtypes of MPS. Hearing loss was found to be common in MPS I, II, III, IVA, VI, and VII, and absent from MPS IVB and MPS IX. MPS VI presents primarily with conductive hearing loss, while the other subtypes  can present with any type of hearing loss . The sensorineural component develops as the disease progresses, but there is no consensus on the etiology of the sensorineural component. Enzyme replacement therapy (ERT) is the most common therapy utilized for MPS, but the effects of ERT on hearing function have been inconclusive. This review highlights a need for more comprehensive and multidisciplinary research on hearing function that includes behavioral testing, objective testing, and temporal bone imaging. This information would allow for better understanding of the progression and etiology of hearing loss. Owing to the prevalence of hearing loss in MPS, early diagnosis of hearing loss and annual comprehensive audiological evaluations are recommended. Mucopolysaccharidoses (MPS) represent a group of rare lysosomal storage disorders. There are currently seven recognized subtypes of MPS caused by a deficiency of one of eleven enzymes involved in the degradation of glycosaminoglycans (GAGs), as shown in Most of the subtypes are caused by autosomal recessive inheritance, with only MPS type II being caused by X-linked recessive inheritance . Both coMPS type I (MPS I) is caused by a deficiency in the lysosomal enzyme alpha-L-iduronidase, which is involved in the degradation of dermatan sulfate (DS) and heparan sulfate (HS) . MPS I wHearing loss is common in both the severe and attenuated phenotypes . ReporteHematopoietic stem cell transplantation (HSCT) and ERT are common treatments for MPS I. HSCT is primarily utilized to treat the severe phenotype, while ERT with laronidase is commonly utilized with the attenuated phenotype . SeveralMPS type II (MPS II), also known as Hunter syndrome OMIM 309900), is the only subtype of MPS that is caused by X-linked recessive inheritance. Males are predominantly affected, but rare female cases have been reported ,55. Hunt9900, is Audiological findings have been well documented in MPS II. An MPS II international registry reported hearing loss data from 554 patients . In a reHunter syndrome presents with components of conductive and sensorineural hearing loss. Patients often first experience conductive hearing loss when they are younger. During childhood, sensorineural components emerge and lead to severe sensorineural hearing loss or mixed hearing loss. Owing to deposits of GAGs in the Eustachian tube and middle ear, frequent otitis media is common in patients with MPS II . AccumulERT has been described as a safe and effective treatment option for patients with MPS II . ERT is MPS type III (MPS III), also known as Sanfilippo syndrome, currently has four recognized subtypes , which are each caused by a deficiency to one of four enzymes that are responsible for the degradation of heparan sulfate, as shown in There is limited research focusing on the assessment of auditory function in human patients with MPS III. Only a handful of studies have reported clinical symptoms of patients with MPS IIIC and MPS IIID ,67,68, aA murine model of MPS IIIB displayed abnormalities in the middle ear, otitis media, hair cell loss in the inner ear, and damage to outer sulcus and pillar cells in the organ of Corti, affecting the base of the cochlea . ChronicCurrently, there are no treatments available for MPS III. Severe impairment of the CNS has been described as a barrier for treatment development . Bone maMPS type IV (MPS IV), also known as Morquio syndrome, is divided into two subtypes; that is, MPS IVA OMIM 253000) and MPS IVB (OMIM 253010). MPS IVA is caused by a deficiency in the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase, which is involved in the degradation of chondroitin-6-sulfate and keratan sulfate (KS) . MPS IVB3000 and Hearing loss is common in MPS IVA ,14,34,35ERT with elosulfase alfa, or Vimizim, is a safe and commonly utilized therapy for patients with MPS IVA ,86,87. WMPS type VI (MPS VI), also known as Maroteaux–Lamy syndrome (OMIM 253200), is a rare autosomal recessive lysosomal storage disorder caused by the deficiency of arylsulfatase B (ARSB) . The redThere are a few studies that have specifically examined hearing issues in MPS VI ,11,14. MThe standard treatment for hearing issues in patients with MPS VI is tympanostomy tube placement to reduce recurrent otitis media . HoweverMPS type VII (MPS VII), also known as Sly syndrome (OMIM 253220), is one of the rarer forms of MPS, with an estimated incidence of 1:300,000 to 1:2,000,000 live births ,41. Sly Murine models of MPS VII have displayed abnormalities in the middle and inner ear. The conductive component has been attributed to cerumen impaction, recurrent otitis media, and ossicle articular alterations . Inner eLimited research was found focusing on the assessment of auditory function in human patients with MPS VII. This may be because of the low incidence and rare nature of Sly syndrome. Recent studies on the audiological assessment of various types of MPS have not included any participants with MPS VII ,11,12,14Murine models have been utilized to observe the efficacity of several therapy methods on the improvement of audiometric results and pathological findings ,44,101. MPS type IX (MPS IX), also known as Natowicz syndrome, is an extremely rare autosomal recessive disorder that causes hyaluronidase deficiency (OMIM 601492). Currently, only four patients have been reported to have MPS IX and the corresponding hyaluronan accumulation . The firUnlike other MPS types, patients with MPS IX do not exhibit hearing loss among the few known reported cases ,46. AlthAlthough there is only a small number of reported cases, animal models can give us insight on varied clinical symptoms and treatment options for patients with MPS IX. MPS IX is caused by the deficiency of hyaluronidase 1 (HYAL1) . Murine This article provides a review of the literature on audiological findings in patients with each of the recognized subtypes of MPS. According to the current literature, hearing loss is common in most of the subtypes of MPS; that is, MPS I (Hurler syndrome), MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IVA (Morquio syndrome type A), MPS VI (Maroteaux–Lamy syndrome), and MPS VII (Sly syndrome). Hearing loss is not present in patients with MPS IVB (Morquio syndrome type B) or MPS IX (Natowicz syndrome), although this is based on a small number of patients owing to the rarity of these subtypes. MPS VI is the only subtype that presents primarily with conductive hearing loss. The other subtypes  can present with any type of hearing loss . In these subtypes, patients often first present with conductive hearing loss and later develop a sensorineural component, leading to sensorineural or mixed hearing loss. It is unclear why MPS VI is the only subtype that does not affect a sensorineural component in general. Sensorineural hearing loss has been reported in some rare cases with MPS VI, although it is not clear whether sensorineural hearing loss is owing to relatively slow progression of MPS VI. While CNS impairment is absent from patients with MPS VI, it is also absent from patients with MPS IVA , a subtyMore comprehensive audiological and pathophysiological research is needed to develop a more precise understanding of the progression of hearing loss in each MPS subtype. In particular, understanding both onsets and etiology of sensorineural hearing loss is critical to provide appropriate clinical intervention to patients. Treatment options vary depending on the MPS subtype. ERT is the most common treatment for MPS, with approved therapies available for ,52, MPS This review highlights the need for more comprehensive audiological research for all subtypes of MPS. Previous studies on hearing loss have primarily utilized behavioral audiological testing and tympanometry. In addition to commonly reported pure tone average threshold levels, future research should report frequency specific hearing thresholds. Frequency specific information  would provide valuable insights into specific cochlear regions affected by MPS. OAE and ABR have occasionally been utilized, but these studies often do not provide detailed results. Temporal bone imaging is rarely utilized in the current literature. In addition to common behavioral audiological assessment, future research should utilize auditory neurophysiological assessment and temporal bone imaging as a part of a comprehensive assessment. A recent paper by van den Broek et al. is a prime example of a comprehensive assessment of auditory function . This reFinally, this review highlights the importance of early diagnosis of hearing loss and annual comprehensive audiological evaluation for patients with MPS. This is consistent with other recommendations found in the literature ,111,112.


Sudden sensorineural hearing loss (SSNHL) is most often defined as a rapid hearing loss of ≥ 30 decibels across at least three contiguous audiometric frequencies over a time of ≤ 72 hours. Cupping therapy has been practiced across the world for thousands of years. Cupping therapy is practiced by creating suction inside cups that are placed on predefined skin areas. Our case is a 48-year-old female with a four-year history of Meniere’s disease, recurrent tinnitus, episodes of dizziness attacks, and fullness of the right ear. The patient developed sudden sensorineural hearing loss. She received conventional treatment and wet cupping therapy as a complementary integrative treatment. After the integrative management protocol was completed, pure tone audiometry tests revealed significant hearing improvement across almost all frequencies. To the best of our knowledge, this case presentation is the first reported case of this type. A positive effect of cupping was reported in our case as an integrative complementary treatment. Large, well-designed quality clinical trials to evaluate the efficacy and safety of wet cupping therapy (WCT) as a complementary treatment of SSNHL is highly recommended. Sudden sensorineural hearing loss (SSNHL) is a hearing loss of ≥ 30 decibels across at least three contiguous audiometric frequencies over a time of ≤ 72 hours . SSNHL cMeniere’s syndrome is a long-term progressive disease that damages the inner ear balance and hearing parts, which may lead to permanent hearing loss . Whilst To date, no studies have examined the effect of cupping therapy on SSNHL or Meniere’s syndrome, despite some studies reporting the effectiveness of using wet cupping therapy (WCT) in the management of various diseases, such as migraine, shoulder pain, and neck pain -7. It alCupping therapy (Al-Hijama in Arabic) means to reduce in size . It has To the best of our knowledge, this case presentation is the first reported case of this type.A 48-year-old female presented with right sudden SSNHL, dizziness, intractable tinnitus, and right ear fullness at our audiology clinic. She had a four-year history of Meniere's disease, recurrent dizziness attacks, an intermittent mild sensation of imbalance usually lasting < 30 minutes, recurrent tinnitus, and fullness of the right ear.Her hearing test showed typical low-frequency SSNHL, reaching up to 45 dB Figure . To idenA full course of prednisolone (1 mg/kg) was prescribed to the patient for two weeks, followed by a tapering dose for another two weeks, together with a diuretics course for seven days and betahistine (16 mg three times per day). The patient then presented to our clinic two weeks later with further deterioration of her hearing by 10-15 dB at low frequencies, reaching up to 60 dB.At this stage, a decision was made to include WCT as a complementary therapy to the treatment protocol after the acceptance of the patient. Written informed consent was obtained from the patient to publish this case report.Cupping therapy was performed by a professional cupping therapist on post-auricular cupping points. A light to medium suction pressure (between 200 and < 350 millibars) was applied for three to five minutes using an electrical suction pump . Other sPure tone audiometry tests revealed a significant hearing improvement by 25-35 dB at low frequencies (reaching to 20 dB) after completion of the WCT treatment regime. Figure Furthermore, the DHI score was improved from an initial 52 score to 0  and the THI score improved 86 to 22 (mild handicap).To the best of our knowledge, there are no published studies that evaluate the effectiveness of WCT in the management of inner ear pathologies or its effect on hearing in SSNHL. Hence, we are unable to compare our findings with other studies. Our presented case  suggested a poor prognosis. SSNHL patients with no improvement in hearing within two weeks are unlikely to recover; those with hearing loss for two to three months are likely to become permanently deaf . Oral coNo local or systemic adverse events were reported in our case report. This is in agreement with previous research where adverse effects or adverse events related to cupping were infrequent, and most adverse effects were avoidable if trained personnel provided cupping therapy .We represent a case of SSNHL treated with WCT and conventional medical treatment. The substantial efficacy of WCT was reported in this case. Adding complementary medicine approaches, such as WCT, to the management protocol of SSNHL is worth trying if routine medical treatments alone fail. Further investigation with large well-designed quality trials to evaluate the efficacy and safety of WCT as a complementary treatment of SSHNL is highly recommended.


High accuracy in estimating energy expenditure is essential for enhancing sports performance. The resting metabolic rate (RMR), as a primary component of total energy expenditure (TEE), is commonly estimated using predictive equations. However, these references may not be applicable to adolescent athletes. The purpose of this cross-sectional study was to analyse the differences between predicted RMR in relation to energy expenditure measured by indirect calorimetry (IC) among 45 Brazilian male adolescent football athletes. Indirect calorimetry (IC) and anthropometric (bioimpedance) measurements were recorded at a single visit to the laboratory after fasting overnight. The mean age was 15.6 ± 1.14 years, body mass was 63.05 ± 7.8 kg, and height was 172 ± 7.5 cm. The RMR values predicted by equations proposed by the Food and Agriculture Organization (FAO) (United Nations), Henry and Rees (HR), Harris Benedict (HB), and Cunningham (CUN) were compared with IC RMR values, by correlation analysis. The FAO and HR predictive equations yielded different values from IC . A moderate correlation of 0.504 was found between the results of HB and IC. In the survival-agreement model, the CUN equation showed low disagreement with the IC RMR, with error values between 200 and 300 kcal/day. The results showed that HB and CUN yielded similar values as IC, with the CUN equation showing low disagreement with IC; hence, adolescent athletes should undergo evaluation with precise laboratory methods to ensure that accurate information about RMR is recorded. To ensure optimal control of body composition and maximal sports performance, it is essential to balance energy intake with energy expenditure. Estimating the total daily energy expenditure (TEE) can be challenging for nutritionists because athletes’ nutritional goals and requirements are not static over the training year .Daily TEE is composed of basal or resting metabolic rate (BMR or RMR), which refers to the energy cost of essential life processes (60–75% of the TEE), diet-induced thermogenesis , and the energy expended during physical activities  , 3. AlthSome of the equations proposed in the literature –7 are coBody composition is an important parameter for football players, who start their competitive careers in adolescence, and from that early age, they are subjected to constant and prolonged physical exhaustion. Football is an endurance sport , charactIn current clinical practice, predictive equations are widely used for estimating the RMR of adolescent athletes . PreviouIn all, 45 male players from the under-15 and under-17 categories of a first division team in the Rio de Janeiro State Association Football Championship participated in this cross-sectional study. The inclusion criteria were training load of at least 4 hours for 5 days each per week, engaged in practice for at least 1 year prior to recruitment, and competed in at least one official national championship season. After club approval and meeting with the coaching committee, a meeting was held with the athletes for recruitment. The participants and their parents or legal guardians were informed about the experimental procedures and possible risks associated with the study, and informed consent was obtained in writing. The participants completed the testing procedures on a single day. Four athletes were evaluated per day for 3 weeks at the Nutrition Evaluation Laboratory of the Federal University of Rio de Janeiro. The football players underwent anthropometric, body composition, and indirect calorimetry measurements after fasting for 8 hours and 24 hours of rest from training. The study was approved by the Research Ethics Committee of the Clementino Fraga Filho University Hospital, Rio de Janeiro Federal University, Brazil (CAAE 58179716.3.0000.5257).Body mass was assessed using a digital scale platform without an anthropometric ruler, Filizola® brand, accurate to 0.05 kg and 150 kg. Height was measured using an Alturexata® portable stadiometer, with a bilateral scale in millimeters (resolution of 1 mm), and field of use of 0.35 to 2.13 m. The participants were asked to remain barefoot for the measurements . The bod2) and carbon dioxide production (VCO2) values were collected by canopy and checked continuously for 30 min. For calculation purposes, the first 10 minutes were discarded to ensure data homogeneity. The VO2 and VCO2 values were used in the equation proposed by Weir [IC was carried out as per Compher’s  protocol by Weir  Eq 1]..2) and cEquations:The following predictive equations were used:The Statistical Program for Social Sciences, version 20.0  was used for statistical analyses. The methods were compared by difference analysis, correlation, degree of agreement, and survival-agreement. Additionally, IC data were also compared against age, anthropometric, and body composition variables to determine associations among these parameters. The Kolmogorov-Smirnov test was used to establish data normality, and the results were expressed as mean and standard deviation. Repeated measures one-way ANOVA was applied to compare mean RMR values between predictive equations and IC at 95% significance (p < 0.05). Pearson’s correlation was used to compare the values from predictive equations; IC values, and the variables of weight, height, age, BMI, FM, and FFM. The coefficients of correlation were considered strong (r > 0.8), moderate (r < 0.8 and >0.5), or weak (r < 0.5). Additionally, the Bland–Altman plot  and survThe characteristics of the participants are shown in The Bland–Altman scatter plots in The agreement-survival results plotted in The Pearson correlations between the body composition variables and IC values were determined. Except for height (r = 0.493) and body mass (r = 0.446), all variables showed moderate correlations to IC values .We aimed to comparatively analyse the suitability of RMR prediction equations and RMR measured by IC in adolescent male football athletes. Our main finding was that the equations yielded RMR values with variations of 200 to 400 kcal from the IC RMR values. The HB and CUN equations showed moderate agreement compared to the FAO and HR equations. These results are in line with previous findings about differences between values obtained from IC and prediction equations .The RMR is the main variable used to calculate daily TEE, so accurate calculation is a crucial requirement . The equAlthough the HB and CUN equations returned better values, the results cannot be considered accurate. Studies conducted with athletes of different modalities have shown that the RMR prediction equations proposed by FAO and HR did not agree with IC RMR values , 23. LouBland–Altman plots were used to present the degree of association between the values obtained from IC and the predictive equations , while sCorrelations between IC and anthropometric parameters were weak or moderate. Sagayama et al.  examinedOur study evaluated whether the equations presented differences between their means in a way that the variation could impair the assessment of a group of athletes. It also verified the agreement between the methods at both the individual and group levels. Finally, we evaluated the disagreement between the methods to verify the margin of error that a practitioner in clinical practice would have to consider when choosing any of the methods.Evaluating FM in adolescent athletes is a challenge in nutritional monitoring . StudiesAnother possible limitation of this study is that nutrient intake was not assessed. Although the athletes were interviewed on food and supplement consumption to ensure RMR protocol compliance, the data were not analysed as nutrient intake. According to Madzima et al. , proteinIn summary, this study evaluated differences between the predicted and IC RMR values. The present results conform with previous findings in the literature on athletes and diverse sports modalities, showing that the use of equations to predict RMR has practical limitations. The predictive equations tested in this study showed differences of 200–400 kcal from the IC RMR values. Therefore, when using predictive equations, the athletes’ energy balance as well as maintenance of body composition, physical performance, and recovery may be compromised. Athletes should undergo RMR evaluation with precise laboratory methods to obtain appropriate dietary prescriptions.The energy needs of football adolescent athletes can be incorrectly estimated using RMR equations. The difference between the RMR values from IC and predictive equations was up to 400 kcal.When choosing a predictive equation to determine an athlete’s RMR, the nutritionist must consider its margin of error and field applicability.S1 File(DOCX)Click here for additional data file.


Single nucleotide polymorphisms in mitochondrial DNA, such as mitochondrial 1555 A>G (m.1555 A>G) and mitochondrial 1494 C>T (m.1494 C>T), are known to be causative mutations of nonsyndromic hearing loss following exposure to aminoglycoside antibiotics. The prevalence of the m.1555 A>G and m.1494 C>T mutations has not been reported for the general population in Japan. The purpose of this study was to investigate the prevalence of m.1555 A>G and m.1494 C>T mutations in a community-dwelling population in Japan in order to prevent aminoglycoside-induced hearing loss. We recruited participants older than 20 years of age to the Iwaki Health Promotion Project in 2014, 2015, and 2016, resulting in the recruitment of 1,683 participants. For each participant, we performed a hearing test and a genetic test for the m.1555 A>G and m.1494 C>T mutations using the TaqMan genotyping method. The m.1555 A>G mutation was detected in only 1 of the 1,683 participants (0.06%). This carrier of the m.1555 A>G mutation was a 69-year-old male with bilateral, symmetric, and high-frequency hearing loss. We provided genetic counseling and distributed a drug card advising him to avoid the administration of aminoglycoside antibiotics. In contrast, the m.1494 C>T mutation was not detected in this study population. Researchers in Japan have measured the prevalence of two hearing loss mutations associated with antibiotic use in the general population. Two mutations in mitochondrial DNA are known to cause hearing loss upon exposure to aminoglycoside antibiotics such as gentamicin. One mutation is found in 3% of Japanese hearing-impaired outpatients, but the frequency in the general population is unknown. A team led by Akira Sasaki of Hirosaki University carried out genetic and hearing tests in 1,683 community-dwelling subjects. They found one person who had one of the mutations. Although this subject had no known history of aminoglycoside antibiotic injection, he exhibited hearing loss at high frequencies. He was provided with genetic counseling and a drug use warning card. The second mutation was not found in the study population. Hearing loss is associated with an array of problems, such as poor quality of life and negative outcomes related to socialization, independence, interpersonal relationships, and communication2. Hearing loss has also been reported to be a significant risk factor for dementia3. In addition, hearing loss is associated with additional costs, including health sector costs, the provision of educational support, the loss of productivity, and societal costs, worldwide1. Thus, hearing loss may result in a significant burden for both individuals and society.The World Health Organization (WHO) estimated that over 5% of the world’s population was affected by hearing loss4. Causative mitochondrial DNA mutations have been found in approximately 5% of patients with postlingual, nonsyndromic hearing loss5. Among the identified mitochondrial mutations, the mitochondrial 1555 A>G (m.1555 A>G) and mitochondrial 1494 C>T (m.1494 C>T) mutations have frequently been identified in patients with aminoglycoside-induced and late-onset, nonsyndromic hearing loss7.Previous studies have shown that hearing loss is a common sensory disorder, affecting 1 in 1000 newborns. A total of 54–68% of hearing loss cases have been estimated to have genetic origins or to be associated with genetic predispositions6. However, no studies have reported the prevalence of the m.1555 A>G and m.1494 C>T mutations in the general population in Japan. It is well known that in carriers of the m.1555 A>G and m.1494 C>T mutations, hearing loss can progress if aminoglycoside antibiotics are administered. Aminoglycoside antibiotics are still being used in many countries, especially for patients with tuberculosis or methicillin-resistant Staphylococcus aureus infection and Neonatal Intensive Care Unit (NICU) babies9.Many reports have described patients with hearing loss who carry these gene mutations. The m.1555 A>G mutation was reported to occur in approximately 3% of Japanese hearing-impaired outpatientsHowever, aminoglycoside-induced hearing loss can be prevented by avoiding the administration of aminoglycoside antibiotics. Therefore, it is very important to investigate the population potentially at high risk for aminoglycoside antibiotic-induced hearing loss.The purpose of this study was to investigate the prevalence of the m.1555 A>G and m.1494 C>T mutations in a community-dwelling population in Japan in order to prevent aminoglycoside-induced hearing loss.10. We invited all residents older than 20 years of age who live in this district to participate in this project. The data collected for this project in 2014, 2015, and 2016 were available for this study.The Iwaki Health Promotion Project is an annual, large-scale, epidemiological survey performed in the Iwaki district of Hirosaki city, JapanData collection and genetic analysis for the present study and the Iwaki Health Promotion Project were approved by the Ethics Committee of Hirosaki University School of Medicine , and all subjects provided written informed consent before participating in the project.Hearing tests were performed for all study participants. Air-conduction, pure-tone thresholds at octave intervals of 0.125, 0.25, 0.5, 1, 2, 4, and 8 kHz were measured by trained examiners using a diagnostic audiometer .13. For the m.1555 A>G mutation, we performed a validation study by using 24 positive control and 24 negative control samples, and for the m.1494 mutation, we performed a validation study with one positive control sample and 24 negative control samples. All TaqMan genotyping results were consistent with the PCR-RFLP, Invader assay, and Sanger sequencing results obtained in our previous studies13.To confirm the accuracy of the TaqMan genotyping method used in this study, we performed a validation study by using positive and negative control DNA samples from our previous cohortWe obtained blood samples from all participants. The presence of the m.1555 A>G and m.1494 C>T mutations was analyzed using the TaqMan genotyping method. Genomic DNA was extracted from 200 µL of a whole-blood specimen using the QIAamp 96 Blood Kit . Then, 0.2 μM m.1494T probe, 0.2 μM m.1494C probe, 0.5μM m.1494CT forward primer and 0.5 μM m.1494CT reverse primer or 0.2 μM m.1555G probe, 0.2 μM m.1555A probe, 0.5 μM m.1555AG forward primer and 0.5 μM m.1555AG reverse primer mixed in SsoAdvanced Supermix  were combined with genomic DNA and analyzed according to the manufacturer’s instructions. Primers and probes were obtained from Eurofins Genomics , and the sequences are presented in Table Approximately 9,500 residents more than 20 years old live in Iwaki district, and 1,167, 1,113, and 1,148 residents participated in this project in 2014, 2015, and 2016, respectively. We excluded overlapping participants, participants without available data, and participants who did not consent to genetic testing. We performed genetic testing for a total of 1,683 subjects , which is slightly lower than the frequency in previous reports. However, this estimate is important as a nonbiased frequency in a certain area where the flow of people is low. Recently, SNP information based on 4,700 healthy volunteers has become available, and the frequency of the m.1555 A>G mutation is found to be 0.15%, which is consistent with our frequency　(https://jmorp.megabank.tohoku.ac.jp/202001/).Approximately 50% of childhood hearing loss is estimated to be associated with genetic contributions, and the prevalence of the m.1555 A>G mutation is 1%37 and 0.01–0.25% among the general population (Table 31. Previous studies reported that the prevalence of the m.1494 C>T mutation was very low, which agreed with the results of this study, in which the m.1494 C>T mutation was not observed among the 1,683 subjects.The m.1494 C>T mutation was not observed in any participants in this epidemiological study. The prevalence of the m.1494 C>T mutation has been reported to be 0.18–0.7% among individuals with hearing loss38. In this study, the audiogram of the subject identified as a carrier of the m.1555 A>G mutation showed a bilateral, symmetric, and high-frequency hearing disorder, which corresponded to the pattern observed for mutation carriers in other studies. In Fig. Some common features of hearing loss have been identified among patients who carry the m.1555 A>G mutation. The audiometric patterns of patients with the m.1555 A>G mutation show bilateral, sensorineural, symmetric, and high-frequency hearing disorders. Hearing loss can also occur either with or without the administration of aminoglycoside antibiotics, and hearing loss can sometimes be progressive. However, hearing loss tends to be severe if aminoglycoside antibiotics have been administered38. Cochlear implantation has also been reported to be an effective therapeutic choice for patients with profound hearing loss caused by this mutation38. Hearing loss has been reported to be a significant risk factor for dementia3, and hearing loss is thought to have functional, social, emotional, and economic impacts. If carriers of m.1555 A>G and m.1494 C>T mutations are administered aminoglycoside antibiotics, then they may suffer from these disadvantages. Therefore, preventing the progression of hearing loss is an important goal. Usami et al. reported that they distributed drug use warning cards not only to patients but also to their relatives to improve avoidance of the administration of aminoglycoside antibiotics14. In our study, we performed genetic counseling and gave the participant a drug use warning card. Despite newer alternative antibiotics, aminoglycoside antibiotics are still being used worldwide9. Thus, the prevention of aminoglycoside administration to m.1555 A>G and m.1494 C>T mutation carriers is important, for example, through the distribution of drug cards.Although hearing loss was relatively mild in the case under study, this subject is at a risk of progressive hearing loss if treated with aminoglycoside antibiotics. No clinically approved treatment has been reported to be effective for sensorineural hearing loss caused by the administration of aminoglycoside antibiotics; however, prescriptive, well-fitted hearing aids can improve hearing among patients with aminoglycoside-induced hearing lossWe were able to identify one m.1555 A>G carrier among a community-dwelling population in the Iwaki district, and we provided genetic counseling and gave him a drug card advising the avoidance of aminoglycoside administration, which will likely contribute to the prevention of hearing loss progression.