Genome sequencing and RNA-seq analyses of mitochondrial complex I deficiency revealed Alu insertion-mediated deletion in NDUFV2.

Isolated complex I deficiency is the most common cause of pediatric mitochondrial disease. Exome sequencing (ES) has revealed many complex I causative genes. However, there are limitations associated with identifying causative genes by ES analysis. In this study, we performed multiomics analysis to reveal the causal variants. We here report two cases with mitochondrial complex I deficiency. In both cases, ES identified a novel c.580G>A (p.Glu194Lys) variant in NDUFV2. One case additionally harbored c.427C>T (p.Arg143*), but no other variants were observed in the other case. RNA sequencing showed aberrant exon splicing of NDUFV2 in the unsolved case. Genome sequencing revealed a novel heterozygous deletion in NDUFV2, which included one exon and resulted in exon skipping. Detailed examination of the breakpoint revealed that an Alu insertion-mediated rearrangement caused the deletion. Our report reveals that combined use of transcriptome sequencing and GS was effective for diagnosing cases that were unresolved by ES.

Coffin-Siris syndrome with bilateral macular dysplasia caused by a novel exonic deletion in ARID1B.

Coffin-Siris syndrome (CSS) is a congenital anomaly syndrome characterized by developmental delay, coarse facial features, and hypoplasia of the fifth digit's nail or phalanges. Herein, we report a case of the 8-year-old female patient who showed developmental delay associated with dysplasia in the macular and large toe area. Comprehensive genomic analysis showed no possible candidate variants, but the subsequent genomic copy number analysis revealed a novel exonic deletion in the coding region of AT-rich interactive domain-containing protein 1B (ARID1B), a gene responsible for CSS. Genomic copy number analysis can aid in diagnosing CSS by confirming undiagnosed exonic deletions in ARID1B. Furthermore, this is the first report of CSS associated with bilateral macular dysplasia.

A diabetic patient in whom Hb Weesp was incidentally detected when her HbA1c level was measured.

The level of glycated hemoglobin A1c (HbA1c) is widely used to monitor long-term glycemic control in patients with diabetes mellitus. There are more than 30 methods for measuring HbA1c levels. In recent times, high-performance liquid chromatography (HPLC) has become the most commonly used method in Japan. However, HPLC-based HbA1c level measurements do not accurately reflect glycemic control in the presence of Hb variants. We report the case of a patient with type 2 diabetes mellitus, who was incidentally found to having an extremely rare Hb variant. A 69-year-old Japanese female visited our clinic and was diagnosed with diabetes mellitus. Her HbA1c level, which was measured using HPLC at our clinic, could not be determined. DNA sequencing revealed a heterozygous mutation in the α1 globin gene (HBA1: c.301C > T, p.Leu101Phe). Hb Weesp was detected. Many Hb variants have been reported; however, to the best of our knowledge, this is only the second report about Hb Weesp in the world and the first from Japan. Clinicians should consider the possibility of Hb variants in cases in which abnormal elution patterns are detected during the measurement of HbA1c using HPLC.

Early onset and focal spike discharges as indicators of poor prognosis for myoclonic-astatic epilepsy.

BACKGROUND: Myoclonic-astatic epilepsy (MAE) is an epileptic syndrome characterized by unique myoclonus, myoclonic-astatic, or astatic seizures in childhood. MAE prognosis vary from spontaneous remission to intractable seizures with profound mental retardation. AIM: Identifying early risk factors may optimize the treatment of children with MAE. Our hypothesis is early onset age and focal spike discharges on EEG indicate a poor MAE prognosis. METHODS: Using the medical records of 9 children with MAE, we analyzed their clinical histories, EEG findings, and seizure symptoms. All patients were given follow-up observations/treatments by our department for at least 2 years after MAE onset. RESULTS: Five of the patients were given favorable prognoses because their seizures disappeared within 2 years of onset; the other 4 received poor prognoses because their seizures continued more than 2 years. MAE onset in patient with refractory seizures was earlier than that in those with a favorable prognosis (7-24 months vs. 23-38 months). All the patients with refractory seizures showed moderate or severe mental retardation. Among the 5 patients with good prognosis, EEGs showed two with focal spike discharges and three with only generalized spike discharges. In contrast, all cases with a poor prognosis had focal spike discharges. CONCLUSIONS: MAE onset in patients with refractory seizures occurs earlier than in those with favorable prognosis. Prognosis was excellent when EEG findings show no focal spike discharges. Both early seizure onset and the focal spike discharges associated with MAE are indicators of poor prognosis.

A Pancreatic Solid Pseudo-Papillary Tumor Detected After Abdominal Injury.

Solid pseudo-papillary tumor (SPT) of the pancreas is a relatively benign tumor that is more frequently reported in females. Most patients usually present with abdominal pain or mass. We experienced the girl who identified SPT with the injury. We diagnosed SPT in a previously healthy 14-year-old Asian girl after abdominal injury. She experienced upper abdominal pain and vomiting after being hit by a basketball. Blood examination revealed a high serum amylase level. Abdominal radiography indicated abnormal bowel gases. Contrast-enhanced computed tomography revealed a smooth, peripheral and unilocular mass approximately 55 mm in diameter in the pancreatic tail. Based on these observations, acute pancreatitis complicated by a pancreatic mass was initially diagnosed. Therapy for acute pancreatitis was instituted, while we simultaneously investigated the mass. Levels of tumor markers were not profoundly elevated in serum. Dynamic contrast-enhanced magnetic resonance imaging (MRI) revealed moderate and gradual increase in contrast-enhanced imaging, consistent with findings of SPT of the pancreas. We thus elected surgical resection for her. Pathological examination of the surgical specimen confirmed our diagnosis of SPT. SPT of the pancreas should be considered as a differential diagnosis of acute abdomen disorders, especially in instances after minor abdominal injuries in young women, and diagnoses must be confirmed with MRIs.

Reduced PLP1 expression in induced pluripotent stem cells derived from a Pelizaeus-Merzbacher disease patient with a partial PLP1 duplication.

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disorder characterized by dysmyelination of the central nervous system (CNS). We identified a rare partial duplication of the proteolipid protein 1 gene (PLP1) in a patient with PMD. To assess the underlying effect of this duplication, we examined PLP1 expression in induced pluripotent stem (iPS) cells generated from the patient's fibroblasts. Disease-specific iPS cells were generated from skin fibroblasts obtained from the indicated PMD patient and two other PMD patients having a 637-kb chromosomal duplication including entire PLP1 and a novel missense mutation (W212C) of PLP1, by transfections of OCT3/4, C-MYC, KLF4 and SOX2 using retro-virus vectors. PLP1 expressions in the generated iPS cells were examined by northern blot analysis. Although PLP1 expression was confirmed in iPS cells generated from two patients with the entire PLP1 duplication and the missense mutation of PLP1, iPS cells generated from the patient with the partial PLP1 duplication manifesting a milder form of PMD showed null expression. This indicated that the underlying effect of the partial PLP1 duplication identified in this study was different from other PLP1 alterations including a typical duplication and a missense mutation.

Diagnosing nocturnal frontal lobe epilepsy: a case study of two children.

We describe two children of nocturnal frontal lobe epilepsy (NFLE) diagnosed using carefully observed nocturnal sleep EEGs and detailed patient histories. Case #1, a 14-year-old boy, showed repeated generalized tonic convulsions and frequent eyes opening seizures during sleep. Conventional EEGs - done with the patient awake or in sleep stage I - showed no abnormalities, while a nocturnal sleep EEG - done during in sleep stage II - revealed the repeated, sharp wave bursts predominantly in the right frontal lobe characteristic of NFLE. During these wave bursts, we noticed the boy's eyes opening, although his parents had not been aware this NFLE symptom. Case #2, a 12-year-old boy, showed one daytime generalized convulsion. He had also been suffering from repeated paroxysmal episodes similar to parasomnia - waking up, sitting, walking, screaming, and speaking - which always followed the same patterns lasting several minutes. During the nocturnal sleep EEG, episodes occurred twice, showing abnormal epileptic discharges predominantly in the frontal lobe. His parents did not mention the episodes to us until questioned, as they had recognized them as parasomnia. The previous conventional EEG showed abnormal slow waves in the frontal lobe, which led us to suspect frontal lobe epilepsy and to take a detailed patient history. The frequency and stereotypy of their symptoms during sleep caused us to perform nocturnal sleep EEGs and led us NFLE diagnosis. Detailed patient histories including sleep habits and carefully observed nocturnal sleep EEGs enabled us to recognize these NFLE clinical features.

Hereditary spherocytosis in 3 children coexisting with UDP-glucuronyl transferase 1A1 deficiency.

Simultaneous presence of hemolytic anemia and bilirubin UDP-glucuronosyltransferase deficiency is a possible cause of misdiagnosis. Seven-year-old and 17-year-old brothers and a 15-year-old sister consecutively suffered from aplastic crises. Although few spherocytes were present, the siblings and their mother had diagnoses of hereditary spherocytosis with flow cytometric analysis of eosin-5'-maleimide-labeled red blood cells in addition to osmotic fragility test. However, inappropriately high values of bilirubin compared with mild hemolysis persisted. Further analysis of UDP-glucuronyltransferase 1A1 revealed all 3 siblings were heterozygous for A(TA)7TAA-P229Q. We report here the importance of careful evaluation of mild hereditary spherocytosis masking UDP-glucuronyltransferase 1A1 deficiency.

[Case of enzyme-linked immunoglobulin with persistent elevation of lactate dehydrogenase activity in serum by administration of an anti-psychotic drug].

We presented a case, who showed extremely high activity of lactate dehydogenase (LD) and confirmed the presence of the LD linked immunoglobulin in her serum. The maximum activity of LD was 6830 IU/L, and the electrophoretic pattern of LD isozymes showed the broad spectrum from isozyme LD2 to LD5. Analysis by counter immuno electrophoresis revealed that immunogloblin was attached to the M subunit of LD and its subtype was an IgG, lambda-chain. The cause, which produced the complex, might be thought to be the side effects by tiapride administrated for her mild dementia. After discontinuance of this drug, the LD activity in serum had gradually reduced. The serum creatinine also increased gradually after administration of tiapride, and did not reverse to normal level by discontinuance of it. The patient died from acute renal failure, which aggravated from sporadic urinary tract infection. It was suggested that her basal renal dysfunction might be due to the LD IgG complexes. We propose a rapid disruption of suspicious drug for the course of the production of LD linked immunoglobulin, because very high titer of these complexes might suffer irreversible damage to the kidney, which chance to become acute renal failure.

[Clinical trial and evaluation of a direct report system of panic values at Fukuoka University Hospital].

In order to support a faster and more informative clinical practice, we established the criteria for panic (critical) values regarding the blood concentrations of glucose, Na, K, Ca, inorganic phosphate (IP), Hb and number of platelets, and also created a system to report these values directly to the doctors in charge. We initiated this system in September 2003. In order to evaluate the availability of this system, we analyzed the clinical data during a one year period, based on the findings of patients showing panic values, mainly concerning the disease states and the correspondences by the doctors who were directly informed. We also carried out questionnaire surveys about the panic values and the new system for all of the doctors in our hospital (recovery rate: 84.3%). The total number of panic values reported was 113 and the mean percentage of the number of ordered examinations was 0.019%. After the report, 79 cases (69.9%) were examined again or treated, while 34 cases (30.1%) had already been treated or watched carefully at the time of the report. Malignant diseases were the main causes of increased panic values (38 cases), especially in the Na, K and blood glucose of patients. The next disease state, which appeared to demonstrate high rates, was chronic renal failure (16 cases), in the low K, high Ca, and low IP patients. Most of the cases of low Hb were caused from bleeding of the gastro-intestinal tract, with malignancies next. A blood infusion was performed for all of the cases with low Hb except for one. As a result of the questionnaire survey among the staff doctors, we confirmed that this system did indeed work efficiently, and 88% of the doctors who answered the questionnaires, were satisfied with the system. In conclusion, we established a new system, which made it possible for panic values to be directly reported to the doctor in charge and this system was then evaluated for its clinical usefulness.

[Diagnosis and gene analysis of congenital hemolytic anemia].

Congenital hemolytic anemia is relatively uncommon and tends to be left undiagnosed because unusual special tests outside health insurance adaptation are needed for the diagnosis. To diagnose properly these patients, we instituted a comprehensive examination system for hemolytic anemia in Fukuoka University Hospital in 1990. In addition to basic laboratory tests, investigation of hemoglobin, SDS-PAGE analysis of red blood cell membrane proteins, a complete panel of red cell enzyme measurements and gene analysis were included. The red cell enzyme measurements was facilitated by the automation on the COBAS MIRA instrument (NIPPON ROCHE). By such a comprehensive examination, we have identified and confirmed 382 cases of congenital hemolytic anemia which otherwise would have escaped detection during the period October 1990 to September 2005 in our institution. SDS-PAGE procedure contributed to the diagnosis of 24 cases of band 3 deficiency, 13 cases of band 4.2 deficiency and 13 cases of band 4.1 deficiency. Hemoglobin studies uncovered 20 cases of abnormal hemoglobins, 126 cases of beta thalassemia and 7 cases of alpha thalassemia. Red cell enzyme measurements were also contributory to the diagnosis of 29 cases of hemolytic anemia in which enzyme levels were reduced.

Porphyromonas gingivalis-induced platelet aggregation in plasma depends on Hgp44 adhesin but not Rgp proteinase.

Evidence from recent epidemiological studies suggests a link between periodontal infections and increased risk of atherosclerosis and related cardiovascular and cerebrovascular events in human subjects. One of the major pathogens of periodontitis, Porphyromonas gingivalis, has the ability to aggregate human platelets in platelet-rich plasma (PRP). Mechanism of P. gingivalis-induced platelet aggregation in PRP was investigated. Proteinase inhibitors toward Arg-gingipain (Rgp) and Lys-gingipain (Kgp) did not suppress P. gingivalis-induced platelet aggregation in PRP, whereas the Rgp inhibitor markedly inhibited P. gingivalis-induced platelet aggregation using washed platelets. Mutant analysis revealed that P. gingivalis-induced platelet aggregation in PRP depended on Rgp-, Kgp- and haemagglutinin A (HagA)-encoding genes that intragenically coded for adhesins such as Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalis-induced platelet aggregation in PRP. P. gingivalis cell-reactive IgG in plasma, and FcgammaRIIa receptor and to a lesser extent GPIbalpha receptor on platelets were found to be a prerequisite for P. gingivalis-induced platelet aggregation in PRP. These results reveal a novel mechanism of platelet aggregation by P. gingivalis.

[Genetic screening for alpha-thalassemia deletional determinants by GapPCR method].

The majority of a-thalassemia results from the large deletions in a-globin gene cluster, including both or either one of alpha-globin genes (alpha1 and alpha2). Most common a-thalassemia-2 deletions (single gene deletions) are -alpha3.7 and -alpha4.2, and alpha-thalassemia-1 deletions (double gene deletions) are --SEA, --THAI, --FIL, --MED and -(alpha)20.5 Although it is not easy to diagnose these deletions because of the high GC content at this locus and the sequence homology among psi alpha2, psi alpha1, alpha2 and alpha1 genes, these alleles can now be diagnosed by a single tube multiplex gapPCR assay. We showed here two Saudi Arabian patients with a-thalassemia trait who could be determined their gene mutations according to the method of Chong SS et al. (2000). [Case 1: 21-year-old male] GapPCR assay revealed the amplification of only -alpha3.7, whereas PCR of both a-globin genes showed no amplifications. The results indicate case 1 is a homozygote of -alpha3.7(-alpha3.7/-alpha3.7). [Case 2: 31-year-old male] GapPCR assay revealed the amplification of only -alpha3.7, and PCR of both alpha globin genes showed normal amplification. DNA sequencing of the amplified a-globin genes revealed a point mutation in the poly A site of alpha2-globin gene (AATAAA-->AATAAG), which is known as alpha(T-Saudi). Thus, case 2 was confirmed to be a compound heterozygote of -alpha3.7 and alpha(T-Saudia) alpha(-alpha3.7 / alpha(T-Saudi) alpha). This gapPCR assay is a rapid, reliable screening test for common alpha-thalassemia deletions and seems to be useful for the diagnosis of thalassemic patients without an increase of Hb A2 and/or an abnormality of beta-globin gene.

[Congenital hemolytic anemia].

Gene analysis in Japanese patients with congenital hemolytic anemia due to red cell membrane disorders, thalassemias, unstable hemoglobinopathies and red cell enzymopathies were summarized. In hereditary spherocytosis, twenty-four mutations of band 3, five mutations of protein 4.2 and twenty mutations of ankyrin have been identified. In beta thalassemia, fourty-seven mutations of beta globin have been found, and ten mutations among them comprise 80% of beta thalassemia patients in Japan. Most common alpha0 and alpha+ thalassemia are--SEA and--alpha3.7, respectively. Fourty glucose-6-phosphate dehydrogenase mutations and twenty-three pyruvate kinase mutations have been identified, allowing a better understanding of the structure-function relationships of these enzymes.

Characterization of a highly polymorphic marker adjacent to the SLC4A1 gene and of kidney immunostaining in a family with distal renal tubular acidosis.

BACKGROUND: Mutations in the human SLC4A1 (AE1/band 3) gene are associated with hereditary spherocytic anaemia and with distal renal tubular acidosis (dRTA). The molecular diagnosis of AE1 mutations has been complicated by the absence of highly polymorphic genetic markers, and the pathogenic mechanisms of some dRTA-associated AE1 mutations remain unclear. Here, we characterized a polymorphic dinucleotide repeat close to the human AE1 gene and performed an immunocytochemical study of kidney tissue from a patient with inherited dRTA with a defined AE1 mutation. METHODS: One CA repeat region was identified in a phage P1-derived artificial chromosome (PAC) clone containing most of the human AE1 gene and the upstream flanking region. We determined its heterozygosity value in multiple populations by PCR analysis. Genotyping of one family with dominant dRTA identified the AE1 R589H mutation, and family member genotypes were compared with the CA repeat length. AE1 and vH(+)-ATPase polypeptides in kidney tissue from an AE1 R589H patient were examined by immunocytochemistry for the first time. RESULTS: This CA repeat, previously reported as D17S1183, is approximately 90 kb upstream of the AE1 gene and displayed considerable length polymorphism, with small racial differences, and a heterozygosity value of 0.56. The allele-specific length of this repeat confirmed co-segregation of the AE1 R589H mutation with the disease phenotype in a family with dominant dRTA. Immunostaining of the kidney cortex from one affected member with superimposed chronic pyelonephritis revealed vH(+)-ATPase-positive intercalated cells in which AE1 was undetectable, and proximal tubular epithelial cells with apparently enhanced apical vH(+)-ATPase staining. CONCLUSIONS: The highly polymorphic dinucleotide repeat adjacent to the human AE1 gene may be useful for future studies of disease association and haplotype analysis. Intercalated cells persist in the end-stage kidney of a patient with familial autosomal dominant dRTA associated with the AE1 R589H mutation. The absence of detectable AE1 polypeptide in those intercalated cells supports the genetic prediction that the AE1 R589H mutation indeed causes dominant dRTA.

Dominant beta-thalassemia with hemoglobin Hradec Kralove: enhanced hemolysis in the spleen.

We describe a 6-year-old girl and her mother with dominant beta-thalassemia due to hemoglobin Hradec Kralove (Hb HK). Both patients presented microcytic anemia, jaundice, splenomegaly, cholelithiasis, and recurrent hemolytic bouts. Osmotic resistance tests using saline and coiled planet centrifugation revealed the increased fragility of the red cell membrane. On the other hand, the glycerol lysing time was prolonged, and results of the isopropanol test were weakly positive. Despite mimicking the features of hereditary spherocytosis, the results of the genetic analyses verified the second reported family with Hb HK (codon 115, GCC [Ala] --> GAC [Asp]). Splenectomy was effective for the amelioration of hemolysis. Of 7 reported patients with Hb variants at beta-globin codon 115 (Hb Madrid and Hb HK), 5 underwent splenectomy. Because of the variable augmentation of extramedullary hemolysis in dominant beta-thalassemias, genotyping is necessary for determining the clinical indication of splenectomy.

Enhanced haemolysis with beta-thalassaemia trait due to the unstable beta chain variant, Hb Gunma, accompanied by hereditary elliptocytosis due to protein 4.1 deficiency in a Japanese family.

We identified a Japanese family with a beta-thalassaemia trait and hereditary elliptocytosis (HE). We studied five members of this family. One was normal, one had only the beta-thalassaemia trait, one had heterozygous HE, and two had compound heterozygous beta-thalassaemia trait and HE. The last two had already undergone splenectomy. The molecular profile of beta-thalassaemia was consistent with that of Hb Gunma: codon 127/128CAGGCT(Gln-Ala)--> CCT(Pro). Analysis of erythrocyte membrane proteins revealed a partial deficiency of protein 4.1 in all those with HE, whereas the spectrin content was within the normal range. Each heterozygous family member with either the beta-thalassaemia trait or HE was asymptomatic, whereas the two with both beta-thalassaemia and HE had marked red blood cell deformities and haemolysis. The abnormalities of the red blood cells in patients with the beta-thalassaemia trait might be enhanced by association with HE owing to a protein 4.1 deficiency.