Real-Time PCR-Based Screening for Homozygous SMN2 Deletion Using Residual Dried Blood Spots.

The survival motor neuron 2 (SMN2) gene is a recognized modifier gene of spinal muscular atrophy (SMA). However, our knowledge about the role of SMN2-other than its modification of SMA phenotypes-is very limited. Discussions regarding the relationship between homozygous SMN2 deletion and motor neuron diseases, including amyotrophic lateral sclerosis, have been mainly based on retrospective epidemiological studies of the diseases, and the precise relationship remains inconclusive. In the present study, we first estimated that the frequency of homozygous SMN2 deletion was ~1 in 20 in Japan. We then established a real-time polymerase chain reaction (PCR)-based screening method using residual dried blood spots to identify infants with homozygous SMN2 deletion. This method can be applied to a future prospective cohort study to clarify the relationship between homozygous SMN2 deletion and motor neuron diseases. In our real-time PCR experiment, both PCR (low annealing temperatures) and blood (high hematocrit values and low white blood cell counts) conditions were associated with incorrect results (i.e., false negatives and positives). Together, our findings not only help to elucidate the role of SMN2, but also aid in our understanding of the pitfalls of current SMA newborn screening programs for detecting homozygous SMN1 deletions.

Newborn Screening for Spinal Muscular Atrophy: A 2.5-Year Experience in Hyogo Prefecture, Japan.

Newborn screening (NBS) for spinal muscular atrophy (SMA) is necessary, as favorable outcomes can be achieved by treatment with disease-modifying drugs in early infancy. Although SMA-NBS has been initiated in Japan, its clinical results have not been fully reported. We report the findings of the initial 2.5 years of a pilot SMA-NBS of approximately 16,000 infants conducted from February 2021 in Hyogo Prefecture, Japan. Clinical data of 17 infants who tested positive were retrospectively obtained from the NBS follow-up centers participating in this multicenter cohort observational study. Genetic testing revealed 14 false positives, and three infants were diagnosed with SMA. Case 1 had two copies of survival motor neuron (SMN) 2 and showed SMA-related symptoms at diagnosis. Case 2 was asymptomatic, with two copies of SMN2. Asymptomatic case 3 had four copies of SMN2 exon 7, including the SMN1/2 hybrid gene. Cases 1 and 2 were treated within 1 month and case 3 at 8 months. All the patients showed improved motor function scores and did not require respiratory support. The identification of infants with SMA via NBS and early treatment improved their motor and respiratory outcomes. Thus, implementation of SMA-NBS at a nationwide scale should be considered.

Improved sensitivity and specificity for citrin deficiency using selected amino acids and acylcarnitines in the newborn screening.

Citrin deficiency is an autosomal recessive disorder caused by a defect of citrin resulting from mutations in the SLC25A13 gene. Intrahepatic cholestasis and various metabolic abnormalities, including hypoglycemia, galactosemia, citrullinemia, and hyperammonemia may be present in neonates or infants in the "neonatal intrahepatic cholestasis caused by citrin deficiency" (NICCD) form of the disease. Because at present, newborn screening (NBS) for citrin deficiency using citrulline levels in dried blood spots (DBS) can only detect some of the patients, we tried to develop a new evaluation system to more reliably detect newborns with citrin deficiency utilizing parameters already in place in present NBS methods. To achieve this goal, we re-analyzed NBS profiles of amino acids and acylcarnitines in 96 NICCD patients, who were diagnosed through selective screening or positive family history. Hereby, we identified the combined evaluation of arginine (Arg), citrulline (Cit), isoleucine+leucine (Ile + Leu), tyrosine (Tyr), free carnitine (C0) / glutarylcarnitine (C5-DC) ratio in DBS as potentially sensitive to diagnose citrin deficiency in pre-symptomatic newborns. In particular, a scoring system using threshold levels for Arg (≥9 µmol/L), Cit (≥ 39 µmol/L), Ile + Leu (≥ 99 µmol/L), Tyr (≥ 96 µmol/L) and C0/C5-DC ratio (≥327) was significantly effective to detect newborns who later developed NICCD, and could thus be implemented in existing NBS programs at no extra analytical costs whenever citrin deficiency is considered to become a novel target disease.

Electrocardiographic R wave amplitude in V6 lead as a predictive marker of cardiac dysfunction in Duchenne muscular dystrophy.

PURPOSE: Duchenne muscular dystrophy (DMD) is an inherited muscular disease characterized by progressive and fatal muscle weakness. Electrocardiographic (ECG) abnormalities, including abnormal R wave amplitudes are frequently observed in DMD. However, clinical implications of abnormal R wave amplitudes remain unclear. Hence, DMD patients were examined for changes in R wave amplitude over time using synthesized 18-lead ECG and the relationship between R wave amplitude and cardiac function. METHODS: The results of 969 ECG examinations of 193 patients with DMD who underwent electrocardiography and echocardiography on the same day were retrospectively reviewed. RESULTS: A negative correlation was observed between R wave amplitude and age. Positive correlations between R wave amplitude and left ventricular ejection fraction were observed in leads V4, V5, V6, syn-V7, syn-V8, and syn-V9, with V6 showing the strongest correlation (r = 0.52). Mean R wave amplitude during cardiac dysfunction was lower than that observed with preserved cardiac function in leads V6 to syn-V9. Patients had preserved R wave amplitude up to three years before the onset of cardiac dysfunction, with a sharp decrease two years before cardiac dysfunction in leads V6 to syn-V9. CONCLUSIONS: In DMD patients, the R wave amplitude decreases with age. The sharp decline in R amplitude two years before cardiac dysfunction indicates that electrophysiological damage to the myocardium of the left ventricle lateral to the posterior wall precedes the finding of cardiac dysfunction. The R amplitude in V6 of the standard 12-lead ECG is a convenient predictive marker of cardiac dysfunction, similar to that of the 18-lead ECG.

Correction: Noguchi et al. PCR-Based Screening of Spinal Muscular Atrophy for Newborn Infants in Hyogo Prefecture, Japan. Genes 2022, 13, 2110.

The authors wish to make the following correction to this paper [...].

Clinical and molecular investigation of 37 Japanese patients with multiple acyl-CoA dehydrogenase deficiency: p.Y507D in ETFDH, a common Japanese variant, causes a mortal phenotype.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is an inherited metabolic disease caused by a defect in electron transfer flavoprotein alpha (ETFA), ETF beta (ETFB), or ETF dehydrogenase (ETFDH), and riboflavin metabolism disorders have recently been reported to present as mimicking MADD. MADD is roughly classified into neonatal (type 1 or 2) and later-onset (type 3) forms. To identify clinicogenetic characteristics in Japan, we investigated 37 Japanese patients with MADD diagnosed from 1997 to 2020. The causes of MADD were ETFDH deficiency in 26 patients, ETFA deficiency in four, ETFB deficiency in six, and riboflavin metabolism disorder in one. All 15 patients with the neonatal-onset type died by 2 years of age, while five of 22 patients with the later-onset form died by 3 years of age. Furthermore, 8 of 15 patients with the later-onset form of ETFDH deficiency treated with riboflavin were riboflavin non-responders. p.Y507D in ETFDH was identified as the most common variant (9 of 48 alleles, 18.8%). Of two patients with a homozygous p.Y507D variant, one experienced disease onset and died in the neonatal period, while the other experienced disease onset at two months of age and died at two years old, suggesting that the p.Y507D variant results in fatal outcomes. Our study concluded that more than half of Japanese patients with MADD died by three years old, and more than half of patients with the later-onset form had poor responsiveness to riboflavin, partly due to the unique Japanese p.Y507D variant in ETFDH.

PCR-Based Screening of Spinal Muscular Atrophy for Newborn Infants in Hyogo Prefecture, Japan.

Spinal muscular atrophy (SMA) is a common devastating neuromuscular disorder, usually involving homozygous deletion of the SMN1 gene. Newly developed drugs can improve the motor functions of infants with SMA when treated in the early stage. To ensure early diagnosis, newborn screening for SMA (SMA-NBS) via PCR-based genetic testing with dried blood spots (DBSs) has been spreading throughout Japan. In Hyogo Prefecture, we performed a pilot study of SMA-NBS to assess newborn infants who underwent routine newborn metabolic screening between February 2021 and August 2022. Hyogo Prefecture has ~40,000 live births per year and the estimated incidence of SMA is 1 in 20,000-25,000 based on genetic testing of symptomatic patients with SMA. Here, we screened 8336 newborns and 12 screen-positive cases were detected by real-time PCR assay. Multiplex ligation-dependent probe amplification assay excluded ten false positives and identified two patients. These false positives might be related to the use of heparinized and/or diluted blood in the DBS sample. Both patients carried two copies of SMN2, one was asymptomatic and the other was symptomatic at the time of diagnosis. SMA-NBS enables us to prevent delayed diagnosis of SMA, even if it does not always allow treatment in the pre-symptomatic stage.

The perioperative transition of serum biomarkers of a 1.5-year-old boy with very-long-chain acyl-CoA dehydrogenase deficiency.

Very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCADD, OMIM 201475) is a congenital fatty acid oxidation disorder. Individuals with VLCADD should avoid catabolic states, including strenuous exercise and long-term fasting; however, such conditions are required when undergoing surgery. The perioperative management of VLCADD in infants has rarely been reported and details regarding the transition of serum biomarkers reflecting catabolic status have not been disclosed. Herein, we present the perioperative clinical and biological data of cryptorchidism in a 1.5-year-old boy with VLCADD. The patient was diagnosed through newborn screening and his clinical course was very stable. Genetic testing of ACADVL revealed compound heterozygous variants c.506 T > C (p.Met169Thr) and c.606-609delC (p.L216*). The enzyme activity of the patient with VLCAD was only 20% compared to that of healthy control. Left orchiopexy for the pediatric cryptorchidism was planned and performed at 1 and a half year of age. Induction anesthesia involved thiopental, fentanyl and rocuronium. The glucose infusion rate was maintained above 6.6 mg/kg/min starting the day before surgery until the operation was completed. Anesthesia was maintained with sevoflurane at approximately 2%. The serum concentration of tetradecenoylcarnitine were stable during the operation, ranging between 0.08 and 0.19 µM (cutoff <0.2 µM), and never deviated from the reference range. Concentration of other serum biomarkers including free fatty acid, 3-OH-butyrate, and creatine kinase, remained similarly unchanged. In this report, we describe the uneventful perioperative management of unilateral orchiopexy for left cryptorchidism in a 1.5-year-old boy with VLCADD using sufficient glucose infusion and volatile anesthesia.

The ACTN3 577XX Null Genotype Is Associated with Low Left Ventricular Dilation-Free Survival Rate in Patients with Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease caused by mutations in the DMD gene. Dilated cardiomyopathy is the leading cause of death in DMD; therefore, further understanding of this complication is essential to reduce morbidity and mortality. METHODS: A common null variant (R577X) in the ACTN3 gene, which encodes α-actinin-3, has been studied in association with muscle function in healthy individuals; however it has not yet been examined in relationship to the cardiac phenotype in DMD. In this study, we determined the ACTN3 genotype in 163 patients with DMD and examined the correlation between ACTN3 genotypes and echocardiographic findings in 77 of the 163 patients. RESULTS: The genotypes 577RR(RR), 577RX(RX) and 577XX(XX) were identified in 13 (17%), 44 (57%) and 20 (26%) of 77 patients, respectively. We estimated cardiac involvement-free survival rate analyses using Kaplan-Meier curves. Remarkably, the left ventricular dilation (> 55 mm)-free survival rate was significantly lower in patients with the XX null genotype (P < 0.01). The XX null genotype showed a higher risk for LV dilation (hazard ratio 9.04). CONCLUSIONS: This study revealed that the ACTN3 XX null genotype was associated with a lower left ventricular dilation-free survival rate in patients with DMD. These results suggest that the ACTN3 genotype should be determined at the time of diagnosis of DMD to improve patients' cardiac outcomes.

False positive cases of elevated tetradecenoyl carnitine in newborn mass screening showed significant loss of body weight.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, a condition in which the body is unable to break down long-chain fatty acids properly, is the most common fatty acid oxidation disorder in Japan. Tandem mass spectrometry has been used in newborn screening (NBS), allowing the detection of patients with VLCAD deficiency even before symptoms manifest. However, tandem mass spectrometry has a high false positive rate. We investigated the clinical characteristics of patients with false positive results for tetradecenoyl acylcarnitine (C14:1). This case-control study used data collected between the 1st of January 2014 and the 31st of March 2019. The case group was defined as patients having levels of both C14:1 and C14:1/C2 ratio higher than cut-off levels in the first newborn mass screening, who were eventually diagnosed as false positives by attending doctors at Kobe University Hospital, Palmore Hospital, or Kakogawa Central City Hospital in Japan. The control group comprised 100 patients randomly selected from the three facilities. The false positive group included 17 cases, and the control group contained 300 patients. The demographics of each group did not show any significant differences in sex, body weight at birth, Cesarean section rate, complete breastfeeding rate, or the number of feedings per day. However, the change in body weight at the sampling day of NBS in the false positive and control groups was -10.2%, and - 4.6%, respectively, showing a statistically significant difference (p < 0.01). In addition, body weight gain at the one-month medical checkup was 38.9 g/day in the false positive group and 44.1 g/day in the control group (p < 0.05). An elevation of C14:1 carnitine has been reported in situations involving the catalysis of fatty acid. Therefore, patients with severe body weight loss might be associated with poor sucking or poor milk supply, which might cause a false positive elevation of C14:1 and C14:1/C2. In suspected VLCAD deficiency, attending doctors should pay attention to body weight changes recorded during newborn mass screening.

Need for strict clinical management of patients with carnitine palmitoyltransferase II deficiency: Experience with two cases detected by expanded newborn screening.

In Japan, carnitine palmitoyltransferase II (CPTII) deficiency has been included as one of the primary target diseases in the expanded newborn mass screening program since 2018. However, many cases of the severe infantile hepatocardiomuscular form of CPTII deficiency showed severe neurodevelopmental delay or sudden death, which indicated that management of CPTII deficiency in the acute phase remains to be studied in detail. Herein, we discuss two cases diagnosed by newborn mass screening. Patient 1 was under strict clinical management from the neonatal period, with >20 admissions in 14 months, while Patient 2 was managed using a relatively relaxed approach, with only 2 admissions in the same period. Patient 1 showed normal development; however, Patient 2 expired at the age of 1 year 2 months. To develop strategies for preventing sudden deaths in patients with CPTII deficiency, this retrospective study focused on detailed clinical management practices and biochemical findings during the acute phase. We also investigated the correlation between conventional biomarkers (such as creatine kinase) and long-chain acylcarnitines. We propose that strict monitoring and immediate medical attention, even in case of slight fever or minor abdominal symptoms, can help prevent sudden death in patients with CPTII deficiency. Considering the higher morbidity rate of such patients, strict and acute management of CPTII deficiency cannot be overemphasized.

Preliminary Effectiveness and Safety of High Frequency Oscillation in Addition to Mechanical Insufflation and Exsufflation for Intratracheal Mucus Removal in Patients With Neuromuscular Disease: Protocol for a Prospective Study.

BACKGROUND: Mechanical insufflation-exsufflation (MI-E) is necessary for noninvasive management of respiratory clearance in patients with neuromuscular disorders (NMDs). Its utility has been proven, and the technique is recommended in a number of international guidelines for the management of patients with NMDs. However, the clearance of thick secretions adhering to the tracheobronchial walls could be problematic when these patients suffer from respiratory tract infections. To improve the effectiveness of the noninvasive technique, a novel device combining MI-E with high frequency oscillation (HFO) has been developed. However, the efficacy of HFO therapy in NMDs has not been well studied. OBJECTIVE: The aim of this study was to elucidate the effect of MI-E combined with HFO for mucus removal in NMD patients. To evaluate its efficacy, changes in transcutaneous oxygen saturation (SpO2), which may predict intratracheal mucus removal, will be measured before and after use of MI-E. METHODS: This is a single-center, nonblinded, nonrandomized prospective study that will enroll 5 subjects hospitalized in Kobe University Hospital owing to respiratory tract infection. All subjects will receive MI-E therapy a few times daily and will receive HFO every other day, for 6 days. Before and after MI-E use, SpO2 will be obtained and the change in SpO2 (ΔSpO2) between MI-E with and without HFO will be calculated. For every subject, the average of ΔSpO2 with or without HFO will be obtained and the null hypothesis that there is a mean change of 0 in the SpO2 between MI-E with and without HFO will be tested using the paired t test. If the treatment with HFO is found to be statistically significantly superior to the treatment without HFO, the study will conclude that HFO addition is more efficacious than no HFO addition. RESULTS: A total of 2 subjects have already been recruited and enrolled in this study as of August 2018. CONCLUSIONS: This unique protocol will assess the efficacy of adding HFO to MI-E during the acute phase of respiratory tract infection in patients with NMDs. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/12102.

The prescription rates of glucagon for hypoglycemia by pediatricians and physicians are low in Japan.

PURPOSE: Hypoglycemia is a common and life-threatening complication in type 1 diabetes mellitus (T1DM) patients. Current guidelines recommend glucagon for treating hypoglycemia in out-of-hospital settings; however, glucagon is reportedly underused in such patients. We conducted a doctor-oriented, questionnaire-based survey of pediatricians and physicians to determine the glucagon prescription rate and identify the reason(s) for its underuse in T1DM patients. METHODS: A questionnaire was mailed to 415 pediatricians and 200 physicians employed at 66 facilities with >100 general wards throughout Hyogo, Japan. The following variables were surveyed: doctor's specialty, glucagon prescription rate, familiarity with glucagon use guidelines, barriers to prescribing glucagon, and attitude changes after education. RESULTS: After 16 doctors were found to have retired, 599 doctors were enrolled; 305 (187 pediatricians and 118 physicians) returned a completed questionnaire. In all, 45 pediatricians and 104 physicians were treating T1DM patients, of whom 24% and 28% reported prescribing glucagon, respectively. The guideline familiarity rate among pediatricians was lower than that among physicians. The major barrier to prescribing glucagon was the complex preparation procedure required by patients/caregivers. More than half of the doctors who did not prescribe glucagon began doing so after being educated about the guidelines. CONCLUSION: The glucagon prescription rate was low among both pediatricians and physicians in Japan.

Renal insufficiency mimicking glutaric acidemia type 1 on newborn screening.

BACKGROUND: Glutaryl carnitine (C5DC) in dried blood spots is used as a biomarker for glutaric aciduria type 1 (GA-1) screening. C5DC, however, is the only screening marker for this condition, and various pathological conditions may interfere with C5DC metabolism. Recently, C5DC elevation has been reported in cases of renal insufficiency. METHOD: Five patients who were positive for GA-1 on newborn screening with tandem mass spectrometry between September 2012 and March 2015 at Kobe University Hospital were enrolled in this study. RESULTS: GA-1 was not confirmed on urinary organic acids analysis in any of the patients. C5DC decreased immediately in four patients, but one patient, who had high C5DC for at least 4 months, was diagnosed with bilateral renal hypoplasia. CONCLUSION: In the case of persistently elevated C5DC, renal insufficiency should be considered as a differential diagnosis.

Newborn screening for carnitine palmitoyltransferase II deficiency using (C16+C18:1)/C2: Evaluation of additional indices for adequate sensitivity and lower false-positivity.

BACKGROUND: Carnitine palmitoyltransferase (CPT) II deficiency is one of the most common forms of mitochondrial fatty acid oxidation disorder (FAOD). However, newborn screening (NBS) for this potentially fatal disease has not been established partly because reliable indices are not available. METHODS: We diagnosed CPT II deficiency in a 7-month-old boy presenting with hypoglycemic encephalopathy, which apparently had been missed in the NBS using C16 and C18:1 concentrations as indices. By referring to his acylcarnitine profile from the NBS, we adopted the (C16+C18:1)/C2 ratio (cutoff 0.62) and C16 concentration (cutoff 3.0nmol/mL) as alternative indices for CPT II deficiency such that an analysis of a dried blood specimen collected at postnatal day five retroactively yielded the correct diagnosis. Thereafter, positive cases were assessed by measuring (1) the fatty acid oxidation ability of intact lymphocytes and/or (2) CPT II activity in the lysates of lymphocytes. The diagnoses were then further confirmed by genetic analysis. RESULTS: The disease was diagnosed in seven of 21 newborns suspected of having CPT II deficiency based on NBS. We also analyzed the false-negative patient and five symptomatic patients for comparison. Values for the NBS indices of the false-negative, symptomatic patient were lower than those of the seven affected newborns. Although it was difficult to differentiate the false-negative patient from heterozygous carriers and false-positive subjects, the fatty acid oxidation ability of the lymphocytes and CPT II activity clearly confirmed the diagnosis. Among several other indices proposed previously, C14/C3 completely differentiated the seven NBS-positive patients and the false-negative patient from the heterozygous carriers and the false-positive subjects. Genetic analysis revealed 16 kinds of variant alleles. The most prevalent, detected in ten alleles in nine patients from eight families, was c.1148T>A (p.F383Y), a finding in line with those of several previous reports on Japanese patients. CONCLUSIONS: These findings suggested that CPT II deficiency can be screened by using (C16+C18:1)/C2 and C16 as indices. An appropriate cutoff level is required to achieve adequate sensitivity albeit at the cost of a considerable increase in the false-positive rate, which might be reduced by using additional indices such as C14/C3.

Clinical and molecular investigation of 14 Japanese patients with complete TFP deficiency: a comparison with Caucasian cases.

Mitochondrial trifunctional protein (TFP) deficiency is an inherited metabolic disorder of mitochondrial fatty-acid oxidation. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is often reported in Caucasian countries due to a common mutation. However, the molecular and clinical basis of complete TFP deficiency has not been extensively reported. In this study, 14 Japanese cases (13 families) with complete TFP deficiency, including 9 previously reported cases, were analyzed to clarify the clinical and molecular characteristics of TFP deficiency. The clinical types of the 14 patients were as follows: 12 cases of neonatal (n=7) or myopathic (n=5) types and 2 cases of intermediate type. Peripheral neuropathy was found in four cases and hypocalcemia due to hypoparathyroidism, which is rarely reported in Caucasian patients, had developed in four cases. Maternal hemolysis, elevated liver enzymes and low platelet count syndrome and acute fatty liver of pregnancy were noted in two and one mothers, respectively. Fourteen mutations were identified in 26 alleles in Japanese patients, including two novel mutations (HADHA: c.361C>T, and HADHA-HADHB: g.26233880_ 26248855del), although no common mutations were found. This study suggests that the molecular and clinical aspects of Japanese patients with TFP deficiencies differ from those of Caucasian patients.

A newborn case with carnitine palmitoyltransferase II deficiency initially judged as unaffected by acylcarnitine analysis soon after birth.

Carnitine palmitoyltransferase II (CPT-2) deficiency, an autosomal recessive disorder of fatty acid oxidation, can be detected by newborn screening using tandem mass spectrometry (TMS). Our case was a boy born at 38 weeks and 6 days of gestation via normal vaginal delivery; his elder sister was affected with CPT-2 deficiency. Acylcarnitine (AC) was analyzed in both dried blood spots (DBS) and serum 2 h after birth to determine whether the boy was also affected. His C16 and C18:1 AC levels in DBS were in the normal range, while his serum long-chain AC levels were marginally increased but lower than those of his sister. After the samples were taken, he was treated with glucose infusion to prevent any catabolism for 2 days. On day 4, the long-chain AC levels in both DBS and serum obtained were higher than those on day 0 and were equivalent to those of his sister. Genetic testing confirmed the presence of the same mutation found in his sister, a homozygous F383Y mutation in the CPT2 gene, thus leading to the diagnosis of CPT-2 deficiency. The sample for TMS should be taken between days 1 and 7. If the sample is not obtained at an appropriate time, correct diagnosis may not be made, as in our case. Although early diagnosis is required, samples taken within 24 h after birth should not be used for TMS.

A de novo 50-bp GNAS Intragenic Duplication in a Patient with Pseudohypoparathyroidism Type 1a.

Germline intragenic mutations in the GNAS locus result in pseudohypoparathyroidism type 1a (PHP1a) and related conditions. Nearly half of the previously reported GNAS intragenic mutations were structural variants, including 3 tandem duplications of 12-25 bp. However, the precise mutation spectrum and the genomic basis of GNAS structural variants remain to be clarified. Here, we report a de novo 50-bp tandem duplication in GNAS (c.723_772dup50, p.Glu259Leufs*29) identified in a patient with typical clinical features of PHP1a. The mutant transcript was predicted to undergo mRNA decay or encode a nonfunctional protein. The 2 breakpoints of the duplication shared a 1-bp microhomology but were not associated with long homology or nucleotide stretches. We also examined the breakpoint structures of 3 previously reported GNAS duplications and found that 1 had a structure similar to that of our case, while the remaining 2 had blunt-ended breakpoints without microhomologies. In silico analyses revealed that the GNAS-flanking region was not enriched with repeats, palindromes, noncanonical DNA motifs, or GC content. This study expands the mutation spectrum of GNAS and provides the first indication that GNAS intragenic structural variants are induced by multiple processes, including nonhomologous end-joining and/or microhomology-mediated break-induced replication, independently of known rearrangement-inducing DNA features.

Efficacy of bezafibrate on fibroblasts of glutaric acidemia type II patients evaluated using an in vitro probe acylcarnitine assay.

INTRODUCTION: We evaluated the effects of bezafibrate (BEZ) on ß-oxidation in fibroblasts obtained from patients with glutaric acidemia type II (GA2) of various clinical severities using an in vitro probe (IVP) assay. METHODS: Cultured fibroblasts from 12 patients with GA2, including cases of the neonatal-onset type both with and without congenital anomalies (the prenatal- and neonatal-onset forms, respectively), the infantile-onset, and the myopathic forms, were studied. The IVP assay was performed by measuring acylcarnitines (ACs) in the cell culture medium of fibroblasts incubated with palmitic acid for 96h in the presence of 0-800µM BEZ using tandem mass spectrometry. RESULTS: The IVP assay showed that 100µM BEZ markedly reduced the level of palmitoylcarnitine (C16) in the neonatal-onset, infantile-onset, and myopathic forms of GA2, either increasing or maintaining a high level of acetylcarnitine (C2), which serves as an index of energy production via ß-oxidation. In the prenatal-onset form, although a small reduction of C16 was also observed in the presence of 100µM BEZ, the level of C2 remained low. At concentrations higher than 100µM, BEZ further decreased the level of ACs including C16, but a concentration over 400µM decreased the level of C2 in most cases. DISCUSSION: BEZ at 100µM was effective for all GA2 phenotypes except for the prenatal-onset form, as a reduction of C16 without deterioration of C2 is considered to indicate improvement of ß-oxidation. The effects of higher doses BEZ could not be estimated by the IVP assay but might be small or nonexistent.