A sensitive and specific diagnostic test for hearing loss using a microdroplet PCR‐based approach and next generation sequencing

Implementing DNA diagnostics in clinical practice for extremely heterogeneous diseases such as hearing loss is challenging, especially when attempting to reach high sensitivity and specificity in a cost‐effective fashion. Next generation sequencing has enabled the development of such a test, but the most commonly used genomic target enrichment methods such as hybridization‐based capture suffer from restrictions. In this study, we have adopted a new flexible approach using microdroplet PCR‐based technology for target enrichment, in combination with massive parallel sequencing to develop a DNA diagnostic test for autosomal recessive hereditary hearing loss. This approach enabled us to identify the genetic basis of hearing loss in 9 of 24 patients, a success rate of 37.5%. Our method also proved to have high sensitivity and specificity. Currently, routine molecular genetic diagnostic testing for deafness is in most cases only performed for the GJB2 gene and a positive result is typically only obtained in 10–20% of deaf children. Individuals with mutations in GJB2 had already been excluded in our selected set of 24 patients. Therefore, we anticipate that our deafness test may lead to a genetic diagnosis in roughly 50% of unscreened autosomal recessive deafness cases. We propose that this diagnostic testing approach represents a significant improvement in clinical practice as a standard diagnostic tool for children with hearing loss. © 2012 Wiley Periodicals, Inc.     

Strumpell's Disease in a Family with Hereditary Focal Segmental Glomerulosclerosis

Strumpell's familial spastic paraplegia is a rare hereditary disease, clinically characterized by progressive disturbance of gait. Focal Segmental Glomerulosclerosis (FSGS) is a frequent glomerulopathy, with an extremely rare familial subtype. The cases of two brothers with Strumpell's disease are reported, who also developed glomerular renal disease, most probably familial FSGS. The genetics of the two disorders, Strumpell's paraplegia and familial FSGS, are discussed, together with the possibility of a parallel transmission.     

DNA Diagnostics of Hereditary Hearing Loss: A Targeted Resequencing Approach Combined with a Mutation Classification System

Although there are nearly 100 different causative genes identified for nonsyndromic hearing loss (NSHL), Sanger sequencing‐based DNA diagnostics usually only analyses three, namely, GJB2, SLC26A4, and OTOF. As this is seen as inadequate, there is a need for high‐throughput diagnostic methods to detect disease‐causing variations, including single‐nucleotide variations (SNVs), insertions/deletions (Indels), and copy‐number variations (CNVs). In this study, a targeted resequencing panel for hearing loss was developed including 79 genes for NSHL and selected forms of syndromic hearing loss. One‐hundred thirty one presumed autosomal‐recessive NSHL (arNSHL) patients of Western‐European ethnicity were analyzed for SNVs, Indels, and CNVs. In addition, we established a straightforward variant classification system to deal with the large number of variants encountered. We estimate that combining prescreening of GJB2 with our panel leads to a diagnosis in 25%–30% of patients. Our data show that after GJB2, the most commonly mutated genes in a Western‐European population are TMC1, MYO15A, and MYO7A (3.1%). CNV analysis resulted in the identification of causative variants in two patients in OTOA and STRC. One of the major challenges for diagnostic gene panels is assigning pathogenicity for variants. A collaborative database collecting all identified variants from multiple centers could be a valuable resource for hearing loss diagnostics.     

Ethinylestradiol and testosterone have divergent effects on circulating IGF system components in adolescents with constitutional tall stature

OBJECTIVE: Pharmacological doses of estrogens or testosterone are used to limit the final height of girls or boys with constitutional tall stature but the mechanism behind this growth inhibition is still debated. We therefore studied the changes in the circulating components of the insulin-like growth factor (IGF) system during high dose sex steroid therapy. DESIGN AND METHODS: Twenty three girls and twenty boys with constitutional tall stature were treated with 100 microg ethinylestradiol per day or 250 mg testosterone ester every 14 days respectively. In 19 girls and 18 boys, the levels of IGF-I, free IGF-I, IGF-II, acid-labile subunit (ALS) and IGF binding proteins (IGFBP)-2 to -6 were measured before and 3-6 months after the start of therapy (group 1). In 18 girls and 11 boys, samples were collected at the end of therapy and 3 to 6 months afterwards (group 2). Fourteen girls and nine boys belonged to both groups. All parameters were measured by radioimmunoassay or ELISA. RESULTS: Levels of IGF-I were decreased significantly by estrogen treatment but remained unchanged during testosterone treatment. Free IGF-I decreased during estrogen treatment but increased during testosterone therapy. Estrogens increased IGF-II and testosterone reduced it. The important reduction of IGFBP-2 during estrogen therapy is not reproduced by androgen therapy, neither is the stimulation by estrogens of IGFBP-4. IGFBP-3 is not modulated by either sex steroid. We found that IGFBP-6 is up-regulated by testosterone but not by estrogens; the reverse is true for ALS, which increased during estrogen treatment but remained unchanged during testosterone treatment. CONCLUSIONS: Our findings demonstrate that androgens and estrogens exert differential effects on the circulating levels of several IGF components.     

Recovery during high-intensity intermittent anaerobic exercise in boys, teens, and men

PURPOSE: This study examined the effects of age on recovery of peak torque of knee extensors (PTEX) and flexors (PTFL), and total work (TW) during high-intensity intermittent 30-s (HI30) and 60-s (HI60) exercise in boys (N=19; age, 11.4+/-0.5 yr), teens (N=17; age, 14.7+/-0.4 yr), and men (N=18; age, 24.1+/-2.0 yr). METHODS: Each age group's subjects were subdivided to participate in an HI30 or an HI60 protocol. The HI30 involved 4x18 maximal knee extensions and flexions (1-min rest between sets), and the HI60 comprised of 2x34 reps (2-min rest). PTEX (N.m.kg), PTFL (N.m.kg), and TW (J.kg) were recorded at each set. The percent recovery of PTEX, PTFL, and TW was calculated as percent of the value achieved in the first set. RESULTS: In HI60, the percent recovery for PTEX, PTFL, and TW after the first set was higher in boys compared with teens and men (P<0.01). In HI30, the percent recovery for PTEX, PTFL, and TW was higher in boys compared with men in all sets (P<0.01), and in teens compared with men in the last two sets (P<0.05). The percent recovery of PTFL and TW was higher in boys compared with teens in the last two sets (P<0.05). Lactate increase was most pronounced in men, less pronounced in teens, and least pronounced in boys (P<0.01). Heart rate recovered faster in boys compared with teens and men in both protocols (P<0.05). CONCLUSIONS: The recovery was faster in boys than in teens and men during HI30 and HI60, as evident by the greater percent recovery in boys for a given time. Furthermore, it appears that the rate of recovery during HI30 and HI60 anaerobic exercise is maturity dependent.     

Fatigue resistance during high-intensity intermittent exercise from childhood to adulthood in males and females

This study examined the maturation pattern of fatigue resistance (FR) from childhood to adulthood in females and males during high-intensity intermittent exercise and compared FR between females and males in childhood and adolescence. Thirty males (boys 11.3 ± 0.5 years, teen-males 14.7 ± 0.3 years, men 24.0 ± 2.1 years) and 30 females (girls 10.9 ± 0.6 years, teen-females 14.4 ± 0.7 years, women 25.2 ± 1.4) participated in this study. They performed high-intensity intermittent exercise (4 × 18 maximal knee flexions and extensions with 1-min rest) on an isokinetic dynamometer at 120°s⁻¹. Peak torque of flexors (PTFL) and extensors (PTEX), and total work (TW) were measured. FR was calculated as % of PTEX, PTFL, and TW in 4th versus 1st set. FR was greater (P < 0.05) in boys versus teen-males and men, and in teen-males versus men. In females, FR was greater (P < 0.05) in girls versus teen-females and women, but not different between teen-females and women. FR was not different in boys versus girls and in teen-males versus teen-females. FR for PTFL, PTEX, and TW correlated negatively (P < 0.001) with the respective peak values (r = −0.68 to −0.84), and FR for TW with peak lactate (r = −0.58 to −0.69). In addition, age correlated (P < 0.01) with FR for males (r = −0.75) and females (r = −0.55). In conclusion, FR during high-intensity intermittent exercise undergoes a gradual decline from childhood to adulthood in males, while in females the adult profile establishes at mid-puberty (14–15 years). The maturation profile of FR in males and females during development appears to reflect the maturation profiles of peak torque, short-term muscle power, and lactate concentration after exercise. T. Tsirini and A. Zafeiridis contributed equally to this work.     

Strumpell's disease in a family with hereditary focal segmental glomerulosclerosis

Strumpell's familial spastic paraplegia is a rare hereditary disease, clinically characterized by progressive disturbance of gait. Focal Segmental Glomerulosclerosis (FSGS) is a frequent glomerulopathy, with an extremely rare familial subtype. The cases of two brothers with Strumpell' s disease are reported, who also developed glomerular renal disease, most probably familial FSGS. The genetics of the two disorders, Strumpell's paraplegia and familial FSGS, are discussed, together with the possibility of a parallel transmission.     

Neuroendocrine tumor markers and enterochromaffin-like cell hyper/dysplasia in type 1 diabetes

OBJECTIVE: Parietal cell antibodies (PCAs) are found in 20% of type 1 diabetic patients, denoting autoimmune gastritis and pernicious anemia, which may predispose to enterochromaffin-like (ECL) cell hyper/dysplasia and gastric carcinoid tumors. We evaluated whether chromogranin A (CgA), 5-hydroxyindole acetic acid (5-HIAA), and neuron-specific enolase (NSE) contribute to screening for ECL cell hyper/dysplasia. RESEARCH DESIGN AND METHODS: Sera from 93 type 1 diabetic patients (53 men and 40 women, 31 PCA(+) and 62 PCA(-), aged 45 +/- 13 years) were analyzed for PCAs by indirect immunofluorescence and for CgA, NSE, and gastrin by radioimmunoassay. Urinary 5-HIAA was tested by high-performance liquid chromatography. Corpus atrophy and ECL cell proliferation were assessed in gastric biopsies. RESULTS: PCA(+) patients had higher gastrin (P < 0.0001) and CgA levels (P = 0.003) and were more prone to autoimmune gastritis (odds ratio [OR] 17, P < 0.0001) and ECL cell hyper/dysplasia (OR = 23, P = 0.005) than PCA(-) subjects. ECL cell hyper/dysplasia was present in seven PCA(+) patients who showed higher CgA levels (P < 0.0001) than subjects without ECL cell hyper/dysplasia, but NSE and 5-HIAA levels were similar. CgA levels correlated with gastrinemia (r = 0.50, P < 0.0001), PCA titer (r = 0.42, P = 0.001), and 5-HIAA levels (r = 0.38, P = 0.012). Logistic regression identified the CgA level (beta = 0.01, P = 0.027) as an independent risk factor for ECL cell hyper/dysplasia when PCA, CgA, 5-HIAA, NSE, gastrin, sex, and age were tested. Multivariate linear regression demonstrated that CgA level was determined by ECL cell density (r = 0.59, P < 0.0001) and gastrin level (r = 0.67, P = 0.02). One PCA(+) patient with elevated gastrin, CgA, and 5-HIAA levels had a gastric carcinoid tumor. CONCLUSIONS: PCA(+) patients, particularly those with high gastrin and CgA levels, risk developing ECL cell hyper/dysplasia. The determination of CgA, but not NSE and 5-HIAA, may complement histology in evaluating ECL cell mass.     

Self-reported substance misuse in Greek male prisoners

The aim of this survey was to determine levels and severity of self-reported alcohol and drug misuse and associated physical and mental health problems in Greek male prisoners. The sample consisted of 80 randomly selected convicted and remanded male prisoners in a prison in northern Greece. The Mini International Neuropsychiatric Interview (MINI) was used to assess psychiatric disorders including substance abuse and dependence. All prisoners who participated completed the Alcohol Use Disorders Identification Test (AUDIT). Those who reported daily use of opiates and stimulants completed the Severity of Dependence Scale (SDS). Information was obtained from medical notes about the prisoners' hepatitis B and HIV status. The MINI identified 27.5% of the prisoners as dependent on opiates, 26.3% on alcohol and 73.8% as cannabis users, while 13.8% were misusing both alcohol and illicit drugs. Severity of dependence was rated, using SDS, as serious for all opiate and stimulant users. In terms of physical health examination of medical records indicated that no prisoner was HIV-positive but 26.5% were hepatitis-B-positive. Of those who had a previous history of substance misuse, 31.2% fulfilled the criteria for depression and 37.5% for antisocial personality disorder. Similarly, 15% of those misusing substances had a previous history of deliberate self-harm and 16% were assessed to have moderate to high suicide risk.