Noninvasive prenatal testing for autosomal recessive conditions by maternal plasma sequencing in a case of congenital deafness.

PURPOSE: The goals of our study were to develop a noninvasive prenatal test for autosomal recessive monogenic conditions and to prove its overall feasibility and potential for clinical integration. METHODS: We recruited a pregnant woman and her spouse, who had a proband child suffering from congenital deafness, and obtained the target-region sequencing data from a semicustom array that used genomic and maternal plasma DNA from three generations of this family. A haplotype-assisted strategy was developed to detect whether the fetus inherited the pathogenic mutations in the causative gene, GJB2. The parental haplotype was constructed using a trio strategy through two different processes, namely, the grandparent-assisted haplotype phasing process and the proband-assisted haplotype phasing process. The fetal haplotype was deduced afterward based on both the maternal plasma sequencing data and the parental haplotype. RESULTS: The accuracy levels of paternal and maternal haplotypes obtained by grandparent-assisted haplotype phasing were 99.01 and 97.36%, respectively, and the proband-assisted haplotype phasing process yielded slightly lower accuracies of 98.73 and 96.79%, respectively. Fetal inheritance of the pathogenic gene was deduced correctly in both processes. CONCLUSION: Our study indicates that the strategy of haplotype-based noninvasive prenatal testing for monogenic conditions has potential applications in clinical practice.

A maternally inherited diabetes and deafness patient with the 12S rRNA m.1555A>G and the ND1 m.3308T>C mutations associated with multiple mitochondrial deletions.

Maternally inherited diabetes and deafness (MIDD) is a mitochondrial syndrome characterized by the onset of sensorineural hearing loss and diabetes in adults. Some patients may have other additional clinical features common in mitochondrial disorders such as pigmentary retinopathy, ptosis, cardiomyopathy, myopathy and renal affections. We report a 40-year-old Tunisian patient presenting maternally inherited type 2 diabetes and deafness (MIDD). A molecular genetic analysis was conducted in the patient and his twin sister, but no reported mutations in the tRNA(Leu(UUR)) and tRNA(Glu) genes were found, especially the two mitochondrial m.3243A>G and the m.14709T>C mutations in muscle and blood leukocytes. The results showed the presence of the mitochondrial NADH deshydrogenase 1 (ND1) homoplasmic m.3308T>C mutation the 2 tested tissues (blood leukocytes and skeletal muscle) of the proband and in the patient's sister blood leukocytes. In addition, we identified the mitochondrial 12S rRNA m.1555A>G mutation in muscle and blood leukocytes. The Long-range PCR amplification revealed the presence of multiple deletions of the mitochondrial DNA extracted from the patient's skeletal muscle removing several tRNA and protein-coding genes. Our study reported a Tunisian patient with clinical features of MIDD in whom we detected the 12S rRNA m.1555A>G and the ND1 m.3308T>C mutations with mitochondrial multiple deletions.

A single-arm, open-label, intervention study to investigate the improvement of glucose tolerance after administration of the 5-aminolevulinic acid (5-ALA) in the patients with mitochondrial diabetes mellitus.

BACKGROUND: Mitochondrial diabetes mellitus (MDM) is characterized by maternal inheritance, progressive neurosensory deafness, insulin secretory disorder, and progressive microvascular complications. Mitochondria are critical organelles that provide energy in the form of adenosine triphosphate (ATP). An impairment of ATP production in pancreatic ß cells is regarded as the main cause of the insulin secretory disorder in patients with MDM, and these patients require insulin replacement therapy early after the diagnosis. The amino acid 5-aminolevulinic acid (5-ALA), a precursor of heme metabolites, is a non-proteinogenic δ amino acid synthesized in mitochondria. An addition of ferrous iron to 5-ALA enhances heme biosynthesis and increases ATP production through an upregulation of the respiratory complex. Several studies have reported that the administration of 5-ALA and ferrous iron to existing treatment improved the glycemic control in both patients with prediabetes and those with type 2 diabetes mellitus. The additional administration of 5-ALA and ferrous iron to MDM patients on insulin therapy may improve their insulin secretory capacity and glycemic control by improving their mitochondrial function. The findings of this study are expected to provide new treatment options for MDM and improve the patients' glycemic control and prognosis. METHODS/DESIGN: This study is a single-arm, open-label pilot intervention study using clinical endpoints to investigate the effects of treatment with 5-ALA plus sodium ferrous citrate (SFC) to patients with MDM on their glucose tolerance. A total of 5 patients with MDM will be administered 5-ALA/SFC (200 mg/d) for 24 weeks. We will perform a 75-g oral glucose tolerance test before and at 24 weeks after the start of this 5-ALA/SFC treatment to evaluate glucose-dependent insulin responses. DISCUSSION: To the best of our knowledge, this study will be the first assessment of the effects of 5-ALA/SFC in patients with MDM. This study will obtain an evidence regarding the effectiveness and safety of 5-ALA/SFC for patients with MDM. TRIAL REGISTRATION: This study was registered with the University Hospital Medical Information Network (UMIN000040581) on July 1, 2020 and with the Japan Registry of Clinical Trials (jRCTs071200025) on August 3, 2020.

Blood homocysteine and folic acid levels may provide reference value for the treatment of sudden total frequency deafness.

BACKGROUND: The cause of sudden sensorineural hearing loss (SSNHL) is still unknown. Literature has indicated that there is a statistically significant correlation between hyperhomocysteinemia and SSNHL, yet there is lack of study in the relationship concerning total frequency deafness subtype of SSNHL. This study investigated the relationship between plasma concentration of homocysteine (Hcy), serum concentration of folic acid and occurrence and treatment responding in total frequency deafness adult patients, and explored whether targeted early intervention was associated with improved clinical outcome in this subgroup. METHODS: A total of 54 consecutive adult patients with diagnosis of sudden total frequency deafness in a single institution was enrolled into the study group. Two control groups were established. Control group 1 was derived from inpatients with normal listening comprehension. Control group 2 included 52 patients with sudden total frequency deafness treated in a parallel hospital. Blood concentration of folic acid and Hcy was investigated. Treatment included Ginkgo biloba extract, dexamethasone, hyperbaric oxygen, folic acid, vitamin B6, and optional vitamin B12. All data was statistically analyzed. Blood level of Hcy and folic acid was compared between study group and control group 1. RESULTS: Although there was no clear evidence for the divergence trend of Hcy and folic acid levels individually, the results showed that the study group had higher blood level of Hcy and lower blood level of folic acid, than control group. In the study group, 24 patients (44.44%) demonstrated treatment effectiveness after the 2-week treatment course. Patients without vertigo had higher effective rate than patients with vertigo (P<0.05). CONCLUSIONS: Effective rate of study group was higher than control group 2 which had no folic acid and vitamin B6/B12 supplement. High blood Hcy and low blood folic acid were closely associated in patients with sudden total frequency deafness. The currently accepted concept of treatment for sudden total frequency deafness is not essentially satisfactory. Testing of plasma Hcy and serum folic acid may provide referential value for its treatment and prognosis evaluation.

Cisplatin-induced hearing loss does not correlate with intracellular platinum concentration.

CONCLUSION: Inductively coupled plasma mass spectrometry (ICP-MS) can be applied to organic tissues obtained from experimental animals. Hearing loss does not correlate with the platinum (Pt) concentration found in the inner ear. Drug structure and affinity to inner ear proteins could explain ototoxicity caused by cisplatin. OBJECTIVES: To analyse Pt affinity for brain and ear tissues (of similar embryologic origin) in the Wistar rat and clearance gradient after a single dose, and to correlate these findings with hearing changes. MATERIALS AND METHODS: Thirty-two Wistar rats were intraperitoneally injected with cisplatin at a dose of 5 mg/kg. Animals were sacrificed after obtaining auditory brain responses (ABRs) at 3, 7, 30 and 90 days (nine, seven, seven and nine animals, respectively). Brain and both temporal bones were extracted from each animal and analysed by ICP-MS to determine the absolute concentrations of the metal. Eight non-treated animals were employed as a control group. RESULTS: The ABR thresholds were significantly elevated in animals from all groups after cisplatin treatment. A maximum accumulation of Pt for inner ear and brain was revealed around the first week: 3.175 (57%) and 0.342 (72%), respectively. Pt significantly accumulated in greater quantities in ear than in brain (p<0.01) and was cleared at a higher rate in brain than in ear (p<0.01) following cochlea/brain ratio analysis. No statistically significant correlation was found between amounts of Pt and hearing loss in the study animals.

Ataxia with vitamin E deficiency associated with deafness.

Ataxia with vitamin E deficiency (AVED) is a rare autosomal recessive disorder, usually with a phenotype resembling Friedreich ataxia, caused by selective impairment of gastrointestinal vitamin E absorption. Vitamin E supplementation improves symptoms and prevents disease progress. In North Africa and Southern Europe, AVED is as common as Friedreich ataxia. There are no reported cases from Turkey. We herein report a 16-year-old Turkish girl with AVED, who was found to have total deletion of the TTPA gene as well as sensorineural deafness, and we present her follow-up data after vitamin E therapy.

Comprehensive Genetic Testing for Deafness from Fresh and Archived Dried Blood Spots.

Comprehensive genetic testing has become integral in the evaluation of children with deafness, but the amount of blood required to obtain DNA can be prohibitive in newborns. Dried blood spots (DBSs) are routinely collected and would provide an alternative source of DNA. Our objective was to evaluate the use of DBSs for comprehensive genetic testing for deafness. DNA derived from fresh and archived DBS samples was compared with DNA from whole blood. We performed next-generation sequencing of all known deafness genes in 4 DBS samples: 2 positive controls, an unknown sample, and a negative control. The DBS-derived DNA was of sufficient quantity and quality for clinical testing. In the 2 positive control samples, pathogenic variants were identified; in the negative control, no pathogenic variants were found; and in the unknown sample, homozygous deletion of the OTOA gene was identified as the cause of deafness. This pilot study shows that comprehensive genetic testing for deafness is feasible with fresh and/or archived DBSs.

Whole mitochondrial genome screening of a family with maternally inherited diabetes and deafness (MIDD) associated with retinopathy: A putative haplotype associated to MIDD and a novel MT-CO2 m.8241T>G mutation.

Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. They can be caused by mutations in both nuclear and mitochondrial DNA. In fact, mitochondrial DNA (mtDNA) defects are known to be associated with a large spectrum of human diseases and patients might present wide range of clinical features with various combinations. Our study reported a Tunisian family with clinical features of maternally inherited diabetes and deafness (MIDD). Accordingly, we performed a whole mitochondrial genome mutational analysis, results revealed a haplotype composed by "A750G, A1438G, G8860A, T12705, T14766C and T16519C", in homoplasmic state, in the mother and transmitted to her daughter and her son. The patient with MIDD2 and retinopathy presented, in addition to this haplotype associated to the MIDD, two de novo variations including a novel one m.8241T>G (p. F219C) in MT-CO2 gene and a known one m.13276G>A (p. M314V) in MT-ND5 gene. The coexistence of these two mutations could explain the retinopathy observed in this patient.

Clinical phenotypes, insulin secretion, and insulin sensitivity in kindreds with maternally inherited diabetes and deafness due to mitochondrial tRNALeu(UUR) gene mutation.

An A-to-G transition in the mitochondrial tRNALeu(UUR) gene at base pair 3243 has been shown to be associated with the maternally transmitted clinical phenotype of NIDDM and sensorineural hearing loss in white and Japanese pedigrees. We have detected this mutation in 25 of 50 tested members of five white French pedigrees. Affected (mutation-positive) family members presented variable clinical features, ranging from normal glucose tolerance (NGT) to insulin-requiring diabetes. The present report describes the clinical phenotypes of affected members and detailed evaluations of insulin secretion and insulin sensitivity in seven mutation-positive individuals who have a range of glucose tolerance from normal (n = 3) to impaired (n = 1) to NIDDM (n = 3). Insulin secretion was evaluated during two experimental protocols: the first involved the measurement of insulin secretory responses during intravenous glucose tolerance test, hyperglycemic clamp, and intravenous injection of arginine. The second consisted of the administration of graded and oscillatory infusions of glucose and studies to define C-peptide kinetics. This protocol was aimed at assessing two sensitive measures of beta-cell dysfunction: the priming effect of glucose on the glucose-insulin secretion rate (ISR) dose-response curve and the ability of oscillatory glucose infusion to entrain insulin secretory oscillations. Insulin sensitivity was assessed by euglycemic-hyperinsulinemic clamp. Evaluation of insulin secretion demonstrated a large degree of between- and within-subject variability. However, all subjects, including those with NGT, demonstrated abnormal insulin secretion on at least one of the tests. In the four subjects with normal or impaired glucose tolerance, glucose failed to prime the ISR response, entrainment of ultradian insulin secretory oscillations was abnormal, or both defects were present. The response to arginine was always preserved, including in subjects with NIDDM. Insulin resistance was observed only in the subjects with overt diabetes. In conclusion, the pathophysiological mechanisms responsible for the development of NIDDM and insulin-requiring diabetes in this syndrome are complex and might include defects in insulin production, glucose toxicity, and insulin resistance. However, our data suggest that a defect of glucose-regulated insulin secretion is an early possible primary abnormality in carriers of the mutation. This defect might result from the progressive reduction of oxidative phosphorylation and implicate the glucose-sensing mechanism of beta-cells.

Studies on the thyroidal defect in an atypical form of Pendred's syndrome.

A 52-yr-old woman and her 47-yr-old brother both had large goiters, short stature, nerve deafness, and mild intellectual impairment. Serum levels of thyroid hormones were normal (sister) on borderline low (brother), urinary 127I levels were normal, and perchlorate discharge tests were negative. After adminstration of 125I, we found normal uptakes and retention of istotope by the thyroid, normal serum half-lives for PB 125I, and absence of labelled iodotyrosines in serum or urine. Particulate fractions from both glands had peroxidase activity by in vitro tests for guaiacol oxidation and tyrosine iodination. Samples from different parts of the sister's gland showed a correlation between peroxidase activity in vitro and radioiodine uptake in vivo. We made three separate preparations of thyroglobulin from the sister and one from the brother by gel filtration on 4% agarose. These were similar to each other and to thyrolobulin samples from normal humans when examined by gel electrophoresis both before and after reduction and alkylation. However, the three thyroglobulins from the same gland differed significantly from one another in their composition of most amino acids. This finding indicates that there can be heterogeneity in the protein portion of thyroglobulin, and since the three samples of thyroglobulin were from the same individual, this heterogeneity was probably not of genetic origin. In these patients we could not demonstrate any of the well-established biochemical defects associated with familial goiter. An abnormality in thyroglobulin structure remains in distinct possibility, but we must defer conclusions until there is more known about thyroglobulin heterogeneity and its relationship to hormone biosynthesis.

Serum thyroid hormone and thyrotropin levels in subjects from endemic goiter regions of New Guinea.

We studied serum thyroid hormone and thyrotropin (TSH) levels in subjects from two regions (Nomane and KarKar) of New Guinea where endemic goiter and/or iodine deficiency are prevalent. The results of the studies in 285 patients from Nomane indicated subnormal serum T4 (mean plus or minus SD, 6.5 plus or minus 2.8 vs 8.4 plus or minus 2.0 mug/1ll ml, for normal Americans, P less than 0.001), supranormal serum T3 (161 plus or minus 51 vs 126 plus or minus 33, ng/100 ml, p less than 0.001), supranormal serum T3/T4 ratio (T3/T4 x 100, 3.1 plus or minus 2.4 vs 1.5 plus or minus 0.4, P less than 0.001) and supranormal serum TSH (16 plus or minus 40 vs 2.7 plus or minus 1.2 muU/ml, P less than 0.001). Serum free T4 and free T3 were measured in 42 subjects. Serum free T4 was subnormal (2.0 plus or minus 0.9 vs 2.8 plus or minus 0.5, ng/100 ml P less than 0.001) and free T3 was elevated (677 plus or minus 150 vs 375 plus or minus 105, pg/100 ml, P less than 0.001). Serum T4 in goitrous patients was significantly lower than that in non-goitrous patients (5.9 vs 6.9 mug/100 ml, P less than 0.005). However, serum T3 and TSH were no different in the presence or absence of goiter. The frequencies of elevated serum TSH or serum T3 in presence of goiter were also no different from those in its absence. The mean values of various thyroid function tests in 37 subjects from KarKar Island were similar to the corresponding values in Nomane subjects. Serum T4, T3 and TSH values in 8 of 13 deaf-mute patients were similar to the corresponding mean values of other inhabitants of the region. However, the remaining 5 patients had deaf-mutism with no appreciable abnormality in thyroid function. Serum TSH correlated inversely with serum T4 (r= -0.31, P less than 0.001). There was, however, no significant relationship between serum TSH and T3 levels. The data suggest that: 1) circulating T4 exerts a significant negative feedback effect on serum TSH level and that this effect of T4 may be even more important than that of circulating T3; 2) factors other than hypothyroidism may be important in the genesis of neurological defects in endemic goiter regions.

Adult T-cell leukemia/lymphoma with initial deafness.

In July 1995, a 43-year-old Japanese man presented with deafness in the right ear. On hospital admission, he had deafness in both ears and right facial palsy. Variously sized lymphoid cells with convoluted nuclei were observed in the cerebrospinal fluid. Surface marker analysis revealed monoclonality of T lymphocytes in the spinal fluid. Similar abnormal cells were observed in peripheral blood and bone marrow. Biopsy specimens of the stomach and prostate showed tight proliferation of large lymphoid cells in the interstitium and epithelium. Antibody against human T-lymphotrophic virus type 1 (HTLV-1) was present. The diagnosis of non-Hodgkin's lymphoma, diffuse type, was made. Seven months later, the patient died of sepsis. Autopsy revealed multiple lymphadenopathy in the abdomen and the infiltration of atypical lymphocytes to the pancreas, kidneys, and other organs. A monoclonal band of HTLV-1 provirus was detected by Southern blot analysis. To our knowledge, this is the first report of adult T-cell leukemia/lymphoma with auditory nerve abnormalities as the initial symptom.

Melatonin induces hyporeactivity caused by type II collagen in peripheral blood lymphocytes from patients with autoimmune hearing losses.

We have studied the behavior of peripheral blood lymphocytes in healthy controls and in patients with various hearing losses. These hearing losses were of an autoimmune origin in which type II collagen and melatonin were either present or absent, activated or not with concanavalin A (Con A). In patients with autoimmune hearing losses, the results showed lymphocytes that displayed hyporeactivity to type II collagen in terms of their proliferative activity in the presence of Con A. The hyporeactivity is specially relevant in those cells which are melatonin incubated. When different nosologic entities were studied, we observed similar lymphocyte hyporeactivity to type II collagen in bilateral sensorineural hearing loss, Ménière's disease and otosclerosis. We conclude that in the lymphocytes of patients with autoimmune hearing losses, there is hyporeactivity to type II collagen when compared to the hyporeactivity of lymphocytes in control groups. This hyporeactivity is revealed when the lymphocytes are activated in the presence of melatonin.

Polycythemia vera associated with Ushers's symdrome.

A 50-year-old man with Usher's syndrome, progressive retinitis pigmentosa, and congenital sensoneural deafness developed polycythemia vera. Usher's syndrome was associated with a variety of congenital herediatry disorders but there was no evidence to support more than a chance association between Usher's syndrome and polycythemia vera.

Capability of serum to convert streptomycin to cytotoxin in patients with aminoglycoside-induced hearing loss.

Individual variations in sensitivity to the ototoxic effects of aminoglycoside antibiotics are well documented. Our research demonstrates that there is an apparent difference in serum from patients who are resistant or susceptible to aminoglycoside ototoxicity. In the first study, the cytotoxicity of sera from patients with and without hearing loss after various time periods following the discontinuation of aminoglycoside treatment was assayed using the isolated outer hair cell toxicity assay. The results indicate that sera from patients with hearing loss were significantly more toxic than sera from patients with normal hearing or minimal hearing loss. This toxicity may persist for up to 1 year after discontinuation of aminoglycoside therapy. In a second study, sera were obtained from patients who had received aminoglycoside therapy several years previously. None of these sera was toxic to isolated outer hair cells in vitro. Streptomycin was then incubated with the sera or a protein fraction isolated from sera, and the incubation mixtures were tested for toxicity. The percentage of damaged outer hair cells was significantly higher when streptomycin had been treated with sera or a serum protein fraction from patients with hearing loss (58+/-10% and 68+/-9%, respectively) than with sera or a serum protein fraction from a control group (10+/-5% and 17+/-4%, respectively). In addition, several incubation mixtures were analyzed using high performance liquid chromatography. A new chromatographic peak was only found in the incubations of streptomycin with serum protein from patients with hearing loss. The results suggest that sera from individuals sensitive to aminoglycoside antibiotics may metabolize these drugs to cytotoxins.

Diabetes mellitus, deafness, muscle weakness and hypocalcemia in a patient with an A3243G mutation of the mitochondrial DNA.

In a 54-year-old woman with diabetes mellitus, hearing loss, muscle weakness and hypocalcemia, caused by idiopathic hypoparathyroidism, an A to G transition at the nucleotide position of 3243 (A3243G mutation) was found in the mitochondrial DNA from her leukocytes. Clinical features of diabetes mellitus and hearing loss in association with the A3243G mutation are compatible with a diagnosis of maternally inherited diabetes and deafness (MIDD). Although hypoparathyroidism is rarely seen in MIDD, we consider that hypoparathyroidism in this patient is a possible phenotype caused by the A3243G mutation of mitochondrial DNA.

Changes in serum osmolarity influence the function of outer hair cells.

Fast motility of outer hair cells (OHC) is thought to be based on a hydromechanic principle. In vitro, the function of OHCs can be disturbed by a change in the osmolarity of the culture medium. Whether changes in the serum osmolarity in vivo can also interfere with OHC motility has not been investigated as yet. Serum osmolarity of New Zealand White rabbits (n = 18) was elevated by a continuous infusion of glucose 40%, decreased by an infusion of aqua dest, or kept constant by an infusion of saline. OHC function was monitored using distortion products of otoacoustic emissions (DPOAE). Input output curves were established between 2 and 5 kHz (geometric mean of f2) with primaries of levels between 35 and 55 dB SPL. Cochlear perfusion was measured using a fluorescence microsphere method. Elevation of the serum osmolarity from 306 +/- 17 mosm/l to 365 +/- 23 induced a decrease of DPOAE between 3 and 12 dB SPL. Cochlear blood flow increased from 0.11 +/- 0.09 to 0.15 +/- 0.10 ml/min/g. When decreasing the serum osmolarity from 303 +/- 9 to 281 +/- 8 mosm/l, only slight changes of the DPOAE could be verified. As in the control group, cochlear perfusion was almost unchanged. In the control group, neither serum osmolarity nor DPOAE changed. Comparable to findings in vitro, increasing the serum osmolarity can lead to a disturbance of OHC function. In patients suffering from sudden hearing loss. dehydration due to physical or mental stress is often observed. This new and promising pathophysiological concept needs further clinical evaluation.

In search of the aetiology of deafness in Nigeria--the probable role of rubella infection.

The present study attempts to establish a relationship between maternal rubella and congenital deafness in Nigeria. For the purpose of the survey, the immunological status with respect to rubella infection of 179 healthy but deaf school children (study group) and 248 school children with normal hearing (control group) was determined. The serological technique employed was the haemagglutination inhibition (HI) test. The percentage of immunized persons ranged from 48.6 to 74.2% in the study group and 55.3 to 68.3% in the control group. Furthermore, the HI antibody titres for the study group were not significantly different from those of the control group although both groups were found to exhibit low HI antibody titres. The presence of low titres of HI antibodies in both groups and the absence of any significant correlation between the state of hearing and HI antibody levels seem to indicate that intrauterine rubella is not an important cause of congenital deafness in Nigeria.

Minimally invasive genetic screen for GJB2 related deafness using dried blood spots.

OBJECTIVE: Nonsyndromic hearing loss is one of the most abundant human sensory disorders, and can be found in 1 out of 1000 newborns. In 60-70% of the cases this disorder is hereditary. The phenotype varies from moderate hearing loss to almost complete deafness, often only revealed in late childhood. Early detection of hearing related genetic variations in the first few weeks of life would allow planning of the audiological and logopedical procedures to maintain the children's normal audiological and speech development, and if required a cochlear implantation can be planned in time. We wanted to evaluate, whether the blood samples collected from neonates onto Guthrie cards (dried blood spots, or DBS), and blood collected from people of various ages into blood collecting tubes is equally usable for genetic testing. The quality of the samples on DBS's for genetic tests after an extended period of storage was evaluated. The methods for sample preparation and analysis were also evaluated. METHODS: Two DNA extraction methods were compared on the samples. We extracted DNA from whole blood with the Versagene Blood Kit from Gentra, and from DBS's with boiling. Allele-specific PCRs (AS-PCR) were carried out on each sample. Samples were analyzed with AS-PCR and sequencing, for the 35delG mutation in the GJB2 (Cx26) gene. Freshly drawn and dried blood spot samples stored for several years were used in the experiments. RESULTS: An AS-PCR method for detecting 35delG mutation on DNA extracted from Guthrie cards was validated. Blood samples up to 10 years of storage were applicable in the screen. 84 patients were found with 35delG mutations, both heterozygous (with no detected hearing related phenotypical discrepancies), and homozygous (phenotipically with moderate to severe hearing loss) forms. CONCLUSIONS: The dried blood spots on Guthrie cards require only three drops of blood to be collected from children, which causes less stress than taking 3 ml of blood. The blood stored on Guthrie cards can be used to store DNA samples for at least 10 years. Even under suboptimal storage conditions the samples' DNA remains intact for genetic testing. Compared to blood collection tubes Guthrie cards cost less, are easier to transport and store.