High-risk screening for Fabry disease in hemodialysis patients in Chiba Prefecture, Japan.

BACKGROUND: High-risk screening for Fabry disease in dialysis patients is an effective means for reducing the number of undiagnosed cases. However, such screening has not been conducted in Chiba Prefecture, Japan. Herein, we aimed to estimate the prevalence of Fabry disease among patients undergoing hemodialysis in Chiba Prefecture by high-risk screening using α-galactosidase A (αGal A) activity measurement, and examine the hemodialysis effect on αGal A activity. METHODS: Patients who underwent maintenance hemodialysis at 25 facilities in Chiba Prefecture were recruited. The αGal A activity was measured using the dried blood spot (DBS) test as the first screening. If the enzyme activity was lower than the cut-off, the second screening was performed with the same method before and after dialysis. RESULTS: Overall, 2924 patients (2036 men and 888 women) were included from which 94 cases (45 men and 48 women) showed decreased αGAL activity in the first screening and 3 (two men and one women) in the second screening. Genetic testing was performed in 3 patients, and the c.1078G > A mutation in GLA gene was detected in one male patient (0.03%). There has been a statistically significant decrease in αGal A activity of DBS at post-dialysis compared to that at pre-dialysis (20.5 ± 10.4 pmol/h/disk and 22.7 ± 11.5 pmol/h/disk, p < 0.0001). CONCLUSION: The prevalence of Fabry disease among patients undergoing hemodialysis in Chiba Prefecture was estimated as 0.03%. This is the first time that dialysis has been shown to affect the αGal A activity.

Early clinical signs and treatment of Menkes disease.

Menkes disease (MD) is an X-linked recessive disorder caused by mutations in ATP7A. Patients with MD exhibit severe neurological and connective tissue disorders due to copper deficiency and typically die before 3 years of age. Early treatment with copper injections during the neonatal period, before the occurrence of neurological symptoms, can alleviate neurological disturbances to some degree. We investigated whether early symptoms can help in the early diagnosis of MD. Abnormal hair growth, prolonged jaundice, and feeding difficulties were observed during the neonatal period in 20 of 69, 16 of 67, and 3 of 18 patients, respectively. Only three patients visited a physician during the neonatal period; MD diagnosis was not made at that point. The mean age at diagnosis was 8.7 months. Seven patients, who were diagnosed in the prenatal stage or soon after birth, as they had a family history of MD, received early treatment. No diagnosis was made based on early symptoms, highlighting the difficulty in diagnosing MD based on symptoms observed during the neonatal period. Patients who received early treatment lived longer than their elderly relatives with MD. Three patients could walk and did not have seizures. Therefore, effective newborn screening for MD should be prioritized.

Guide for diagnosis and treatment of hyperphenylalaninemia.

IMPORTANCE: Sapropterin hydrochloride, a natural coenzyme (6R-tetrahydrobiopterin) of phenylalanine hydroxylase, was first approved as a treatment for tetrahydrobiopterin deficiency in 1992 in Japan, and was then approved as a treatment for a tetrahydrobiopterin-responsive hyperphenylalaninemia in 2007 and 2008, in the USA and Japan, respectively. Guidelines are required on the proper use of sapropterin hydrochloride for tetrahydrobiopterin-responsive hyperphenylalaninemia. OBSERVATIONS: It is recommended that tetrahydrobiopterin-responsive hyperphenylalaninemia should be diagnosed in all cases of hyperphenylalaninemia, including phenylketonuria, by tetrahydrobiopterin administration tests rather than by phenotype or blood phenylalanine levels. CONCLUSIONS AND RELEVANCE: If tetrahydrobiopterin-responsive hyperphenylalaninemia is diagnosed, all ages can be treated with sapropterin hydrochloride. Although there are reports that sapropterin hydrochloride is effective and safe for the prevention of maternal phenylketonuria, further investigation is required.

Efficacy of bezafibrate in two patients with mitochondrial trifunctional protein deficiency.

Mitochondrial trifunctional protein (TFP) deficiency is a rare inherited metabolic disorder caused by defects in fatty acid ß-oxidation (FAO) of long-chain fatty acids, leading to impaired energy production. Fasting avoidance, fatty acid-restricted diets, and supplementation with medium-chain triglycerides are recommended as a treatment, but there are no pharmaceutical treatments available with strong evidence of efficacy. Bezafibrate, which enhances the transcription of FAO enzymes, is a promising therapeutic option for FAO disorders (FAODs). The effectiveness of bezafibrate for FAODs has been reported in some clinical trials, but few clinical studies have investigated its in vivo efficacy toward TFP deficiency. Herein, we describe two Japanese patients with TFP deficiency. Patient 1 presented with recurrent myalgia since the age of 5 years. Laboratory findings showed increased serum levels of long-chain fatty acids and reduced expression of TFPα and TFPß in his skin fibroblasts. Based on these findings, he was diagnosed with the myopathic type of TFP deficiency. Patient 2 suddenly exhibited cardiopulmonary arrest one day after birth. Elevated levels of creatine kinase and long-chain acylcarnitines were observed. Genetic analysis identified compound heterozygous variants in HADHB (c.1175C>T/c.1364T>G). He was diagnosed with the lethal type of TFP deficiency. Although both patients were treated with dietary therapy and l-carnitine supplementation, they experienced frequent myopathic attacks associated with respiratory infections and exercise. After the initiation of bezafibrate, their myopathic manifestations were markedly reduced, leading to an improvement in quality of life without any side effects. Our clinical findings indicate that bezafibrate combined with other treatments such as dietary therapy may be effective in improving myopathic manifestations in TFP deficiency.

A simple method for sequencing the whole human mitochondrial genome directly from samples and its application to genetic testing.

Next-generation sequencing (NGS) is a revolutionary sequencing technology for analyzing genomes. However, preprocessing methods for mitochondrial DNA (mtDNA) sequencing remain complex, and it is required to develop an authenticated preprocessing method. Here, we developed a simple and easy preprocessing method based on isothermal rolling circle mtDNA amplification using commercially available reagents. Isothermal amplification of mtDNA was successfully performed using both nanoliter quantities of plasma directly and 25 ng of total DNA extracted from blood or tissue samples. Prior to mtDNA amplification, it was necessary to treat the extracted total DNA with Exonuclease V, but it was not required to treat plasma. The NGS libraries generated from the amplified mtDNA provided sequencing coverage of the entire human mitochondrial genome. Furthermore, the sequencing results successfully detected heteroplasmy in patient samples, with called mutations and variants matching those from previous, independent, Sanger sequencing analysis. Additionally, a novel single nucleotide variant was detected in a healthy volunteer. The successful analysis of mtDNA using very small samples from patients is likely to be valuable in clinical medicine, as it could reduce patient discomfort by reducing sampling-associated damage to tissues. Overall, the simple and convenient preprocessing method described herein may facilitate the future development of NGS-based clinical and forensic mtDNA tests.

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 genetic features of lysinuric protein intolerance in Japan.

BACKGROUND: Lysinuric protein intolerance (LPI) is a rare autosomal recessive disorder affecting the transport of cationic amino acid caused by mutations in solute carrier family 7 amino acid transporter light chain, y+ L system, member 7 (SLC7A7). This disorder occurs worldwide, especially in Finland and Japan, where founder effect mutations have been reported. Detailed features of the clinical symptoms and mutation types in Japanese LPI, however, remain unclear to date. METHODS: An epidemiological nationwide survey of LPI patients was carried out via mail to all domestic university and general hospitals in Japan. Next, the clinical information for each LPI patient was obtained, in the form of a questionnaire, from the attending physicians who replied to the letters. RESULTS: We received answered questionnaires for 43 LPI patients in 19 hospitals. We selected 35 patients who were genetically diagnosed with LPI. The most common clinical manifestations were with protein aversion, ferritinemia, increased serum lactate dehydrogenase, and hyperammonemia. The most frequent SLC7A7 mutation in Japanese LPI patients is p.R410*, which is a founder effect mutation in northern Japan. In total, nine types of mutation were detected in this survey, six of which (p.R410*, p.S238F, c.1630delC, p.S489P, c.1673delG, and IVS3-IVS5del9.7 kb) have not been reported in other countries. CONCLUSION: The clinical and genetic features of 35 Japanese patients with LPI were characterized, and no correlation between genotype and phenotype was observed. The importance of early diagnosis for better prognosis of LPI is emphasized.

Drugs indicated for mitochondrial dysfunction as treatments for acute encephalopathy with onset of febrile convulsive status epileptics.

We studied the efficacy of drugs indicated for mitochondrial dysfunction in the treatment of 21 patients with acute encephalopathy with onset of febrile convulsive status epilepticus at our hospital from January 2006 to December 2014. Among them, 11 patients had been treated with a mitochondrial drug cocktail consisting of vitamin B1, vitamin C, biotin, vitamin E, coenzyme Q10, and l-carnitine (prescription group) and 10 patients were not treated with the cocktail (non-prescription group). We retrospectively reviewed age, trigger, clinical form, treatment start time, and sequelae. Clinical form was classified into a biphasic group presenting acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) and a monophasic group. Sequelae were classified as (A) no sequelae group or (B) sequelae group, and differences in the interval between diagnosis and treatment were also evaluated. The sequelae were not different between the mitochondrial drug cocktail prescription and non-prescription groups, but significantly better in the group administered the mitochondrial drug cocktail within 24h (P=0.035). We expect that early treatment with a mitochondrial drug cocktail could prevent sequelae in acute encephalopathy with onset of febrile convulsive status epilepticus.

Inborn errors of ketone body utilization.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency and mitochondrial acetoacetyl-CoA thiolase (beta-ketothiolase or T2) deficiency are classified as autosomal recessive disorders of ketone body utilization characterized by intermittent ketoacidosis. Patients with mutations retaining no residual activity on analysis of expression of mutant cDNA are designated as severe genotype, and patients with at least one mutation retaining significant residual activity, as mild genotype. Permanent ketosis is a pathognomonic characteristic of SCOT-deficient patients with severe genotype. Patients with mild genotype, however, may not have permanent ketosis, although they may develop severe ketoacidotic episodes similar to patients with severe genotype. Permanent ketosis has not been reported in T2 deficiency. In T2-deficient patients with severe genotype, biochemical diagnosis is done on urinary organic acid analysis and blood acylcarnitine analysis to observe characteristic findings during both ketoacidosis and non-episodic conditions. In Japan, however, it was found that T2-deficient patients with mild genotype are common, and typical profiles were not identified on these analyses. Based on a clinical study of ketone body utilization disorders both in Japan and worldwide, we have developed guidelines for disease diagnosis and treatment. These diseases are treatable by avoiding fasting and by providing early infusion of glucose, which enable the patients to grow without sequelae.

Intracellular in vitro probe acylcarnitine assay for identifying deficiencies of carnitine transporter and carnitine palmitoyltransferase-1.

Mitochondrial fatty acid oxidation (FAO) disorders are caused by defects in one of the FAO enzymes that regulates cellular uptake of fatty acids and free carnitine. An in vitro probe acylcarnitine (IVP) assay using cultured cells and tandem mass spectrometry is a tool to diagnose enzyme defects linked to most FAO disorders. Extracellular acylcarnitine (AC) profiling detects carnitine palmitoyltransferase-2, carnitine acylcarnitine translocase, and other FAO deficiencies. However, the diagnosis of primary carnitine deficiency (PCD) or carnitine palmitoyltransferase-1 (CPT1) deficiency using the conventional IVP assay has been hampered by the presence of a large amount of free carnitine (C0), a key molecule deregulated by these deficiencies. In the present study, we developed a novel IVP assay for the diagnosis of PCD and CPT1 deficiency by analyzing intracellular ACs. When exogenous C0 was reduced, intracellular C0 and total AC in these deficiencies showed specific profiles clearly distinguishable from other FAO disorders and control cells. Also, the ratio of intracellular to extracellular C0 levels showed a significant difference in cells with these deficiencies compared with control. Hence, intracellular AC profiling using the IVP assay under reduced C0 conditions is a useful method for diagnosing PCD or CPT1 deficiency.

Impact of enzyme activity assay on indication in liver transplantation for ornithine transcarbamylase deficiency.

There are no objective and concrete guidelines for the management of Ornithine transcarbamylase deficiency (OTCD). Based on previous findings, we hypothesized that patients with OTCD have a low Ornithine transcarbamylase (OTC) activity in the liver, and therefore it would be better to determine the appropriate indications and optimal timing for liver transplantation (LT) based on the OTC activity. However, few data have so far been accumulated on the OTC activity in cases that are indicated for LT. The purpose of the present study was to clarify the OTC activity in cases that were indicated for LT. This study involved thirteen children with OTCD (8 males and 5 females) who underwent LT, and two females with OTCD who did not require LT. The OTC activity of the neonatal onset type ranged from 0% to 7.2%, while that of the late onset type who underwent LT ranged from 4.4% to 18.7%. The OTC activity of the late onset type which did not require LT was 33-38% based on a preoperative needle liver biopsy. Some late onset patients that underwent LT, showed an activity that was as low as that observed in the neonatal onset cases. This is the first report to show the results of measuring the OTC activity for serial OTCD cases indicated for LT. OTC activity might be an indicator to determine the indications for and the timing of LT in the late onset type, however, further investigations are necessary.

Long-term outcome and intervention of urea cycle disorders in Japan.

Urea cycle disorders (UCDs) are one of the most frequently inherited metabolic diseases in Japan, with an estimated prevalence of 1 per 50,000 live births. Here, we investigated the clinical manifestations, treatment, and prognosis of 177 patients with UCDs who were evaluated and treated from January 1999 to March 2009. These included 77 cases of neonatal-onset UCDs and 91 cases of late-onset UCDs. The most common UCD was ornithine transcarbamylase deficiency (OTCD), which accounted for 116 out of 177 patients. This result is similar to a previous study performed between 1978 and 1995 in Japan: OTCD accounted for about two-thirds of the total number of UCD cases. We studied the relationship between prognosis and the peak blood ammonia level at the onset in 151 UCD patients. Compared with a previous survey conducted in Japan, we found that a greater number of patients survived without any mental retardation despite their peak blood ammonia levels being greater than 360 µmol/l. The 5-year survival rate of patients with OTCD improved to 86% for those with the neonatal-onset type and to 92% for those with the late-onset type. We hypothesize that the increased survival rate is due to early diagnosis and better treatments that are now available in Japan. It is very important to diagnose and treat UCDs, especially OTCD, when the blood ammonia levels in patients are low. The outcome in patients with low blood ammonia levels was better than that in patients with high blood ammonia levels.

Cross-sectional study of bone metabolism with nutrition in adult classical phenylketonuric patients diagnosed by neonatal screening.

The mechanism underlying the development of osteopenia or osteoporosis in longstanding phenylketonuria (PKU) remains to be clarified. We investigated the details of bone metabolism in 21 female and 13 male classical PKU patients aged 20-35 years. Vitamin D (VD), parathyroid hormone (PTH), bone turnover markers, and daily nutrient intake were examined. The patients had lower daily energy and protein intake than did the age-matched controls (22 women, 14 men), but their respective fat, VD, and calcium intake did not differ. Serum 1,25-dihydroxy VD and 25-hydroxy VD levels in female and male patient groups were significantly higher and lower than those in respective control groups (females, P < 0.001; males, P < 0.05 and P < 0.01, respectively). Serum intact PTH levels were significantly higher in the female patient group (P < 0.05). Urinary calcium levels in the patient groups were significantly higher than those of the control subjects (females, P < 0.001; males, P < 0.05). Bone resorption markers were significantly higher in patients than in controls, although bone formation markers were not different. Patient serum levels of osteoprotegerin-inhibiting bone resorption were significantly lower (females, P < 0.001; males, P < 0.01). None of the bone parameters correlated significantly with serum phenylalanine or nutrient intake. PKU patients exhibited lower VD status and more rapid bone resorption despite normal calcium-VD intakes.

Experimental evidence that phenylalanine is strongly associated to oxidative stress in adolescents and adults with phenylketonuria.

Few studies have looked at optimal or acceptable serum phenylalanine levels in later life in patients with phenylketonuria (PKU). This study examined the oxidative stress status of adolescents and adults with PKU. Forty PKU patients aged over fifteen years were enrolled, and were compared with thirty age-matched controls. Oxidative stress markers, anti-oxidant enzyme activities in erythrocytes, and blood anti-oxidant levels were examined. Nitric oxide (NO) production was also examined as a measure of oxidative stress. Plasma thiobarbituric acid reactive species and serum malondialdehyde-modified LDL levels were significantly higher in PKU patients than control subjects, and correlated significantly with serum phenylalanine level (P<0.01). Plasma total anti-oxidant reactivity levels were significantly lower in the patient group, and correlated negatively with phenylalanine level (P<0.001). Erythrocyte superoxide dismutase and catalase activities were higher and correlated significantly with phenylalanine level (P<0.01). Glutathione peroxidase activity was lower and correlated negatively with phenylalanine level (P<0.001). The oxidative stress score calculated from these six parameters was significantly higher in patients with serum phenylalanine of 700-800 µmol/l. Plasma anti-oxidant substances, beta-carotene, and coenzyme Q(10) were also lower (P<0.001), although the decreases did not correlate significantly with the phenylalanine level. Serum nitrite/nitrate levels, as stable NO products, were higher together with low serum asymmetric dimethylarginine, as an endogenous NO inhibitor. Oxidative stress status is closely linked with serum phenylalanine levels. Phenylalanine level in should be maintained PKU below 700-800 µmol/l even in adult patients.

[Acute encephalopathy in inherited metabolic diseases].

Acute encephalopathy, regardless of the cause, is a medical emergency. In addition to being a common manifestation of a variety of acquired medical or surgical conditions, it is a presenting feature of number of inherited metabolic diseases, particularly in young children. Because of the importance of identifying treatable inherited metabolic diseases early, initial investigation must not be delayed. In addition, it is important to keep their serum and urine for investigation later.

A neonatal-onset succinyl-CoA:3-ketoacid CoA transferase (SCOT)-deficient patient with T435N and c.658-666dupAACGTGATT p.N220_I222dup mutations in the OXCT1 gene.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency causes episodic ketoacidotic crises and no apparent symptoms between them. Here, we report a Japanese case of neonatal-onset SCOT deficiency. The male patient presented a severe ketoacidotic crisis, with blood pH of 7.072 and bicarbonate of 5.8 mmol/L at the age of 2 days and was successfully treated with intravenous infusion of glucose and sodium bicarbonate. He was diagnosed as SCOT deficient by enzymatic assay and mutation analysis. At the age of 7 months, he developed a second ketoacidotic crisis, with blood pH of 7.059, bicarbonate of 5.4 mmol/L, and total ketone bodies of 29.1 mmol/L. He experienced two milder ketoacidotic crises at the ages of 1 year and 7 months and 3 years and 7 months. His urinary ketone bodies usually range from negative to 1+ but sometimes show 3+ (ketostix) without any symptoms. Hence, this patient does not show permanent ketonuria, which is characteristic of typical SCOT-deficient patients. He is a compound heterozygote of c.1304C > A (T435N) and c.658-666dupAACGTGATT p.N220_I222dup. mutations in the OXCT1 gene. The T435N mutation was previously reported as one which retained significant residual activity. The latter novel mutation was revealed to retain no residual activity by transient expression analysis. Both T435N and N220_I222 lie close to the SCOT dimerization interface and are not directly connected to the active site in the tertiary structure of a human SCOT dimer. In transient expression analysis, no apparent interallelic complementation or dominant negative effects were observed. Significant residual activity from the T435N mutant allele may prevent the patient from developing permanent ketonuria.

Carnitine palmitoyltransferase 2 deficiency: the time-course of blood and urinary acylcarnitine levels during initial L-carnitine supplementation.

Carnitine palmitoyltransferase 2 (CPT2) deficiency is one of the most common mitochondrial beta-oxidation defects. A female patient with an infantile form of CPT2 deficiency first presented as having a Reye-like syndrome with hypoglycemic convulsions. Oral L-carnitine supplementation was administered since serum free carnitine level was very low (less than 10 micromol/L), indicating secondary carnitine deficiency. Her serum and urinary acylcarnitine profiles were analyzed successively to evaluate time-course effects of L-carnitine supplementation. After the first two days of L-carnitine supplementation, the serum level of free carnitine was elevated; however, the serum levels of acylcarnitines and the urinary excretion of both free carnitine and acylcarnitines remained low. A peak of the serum free carnitine level was detected on day 5, followed by a peak of acetylcarnitine on day 7, and peaks of long-chain acylcarnitines, such as C16, C18, C18:1 and C18:2 carnitines, on day 9. Thereafter free carnitine became predominant again. These peaks of the serum levels corresponded to urinary excretion peaks of free carnitine, acetylcarnitine, and medium-chain dicarboxylic carnitines, respectively. It took several days for oral L-carnitine administration to increase the serum carnitine levels, probably because the intracellular stores were depleted. Thereafter, the administration increased the excretion of abnormal acylcarnitines, some of which had accumulated within the tissues. The excretion of medium-chain dicarboxylic carnitines dramatically decreased on day 13, suggesting improvement of tissue acylcarnitine accumulation. These time-course changes in blood and urinary acylcarnitine levels after L-carnitine supplementation support the effectiveness of L-carnitine supplementation to CPT2-deficient patients.

A novel mutation (c.951C>T) in an exonic splicing enhancer results in exon 10 skipping in the human mitochondrial acetoacetyl-CoA thiolase gene.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inherited disorder affecting isoleucine catabolism and ketone body metabolism. A Japanese female developed a severe ketoacidotic attack at the age of 7 months. Urinary organic acid analysis showed elevated excretion of 2-methyl-3-hydroxybutyrate but not tiglylglycine. She was diagnosed as having T2 deficiency by enzyme assay using fibroblasts. Mutation analysis revealed a compound heterozygote of c.556G>T(D186Y) and c.951C>T(D317D). Since c.951C>T does not cause amino acid change, we performed cDNA analysis and found that exon 10 skipping had occurred in the c.951C>T allele. A computer search using an ESE finder showed that an exonic splicing enhancer sequence, SF2/ASF, was located in CTGA(951)CGC. We hypothesized that the exonic splicing enhancer is necessary for accurate splicing since the first nucleotide of exon 10 is C, which weakens the splice acceptor site of intron 9. We made a mini gene construct including exon 9-truncated intron 9-exon 10-truncated intron 10-exon 11 for a splicing experiment. We also made three mutant constructs which alter the SF2/ASF site (947C>T, 951C>T, 952G>A). An min-gene splicing experiment clearly showed that exon 10 skipping was induced in all three mutant constructs. Moreover, additional substitution of G for C at the first nucleotide of exon 10 resulted in normal splicing in these three mutants. These results confirmed that c.951C>T diminished the effect of the exonic splicing enhancer and caused exon 10 skipping.

Histological findings in the livers of patients with neonatal intrahepatic cholestasis caused by citrin deficiency.

AIM: To characterize the histological features of the livers of patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), we studied specimens from 30 patients diagnosed with NICCD by genetically analyzing the SLC25A13 gene. METHODS: Liver biopsy specimens were subjected to hematoxylin-eosin, Azan, and Berlin-blue staining. RESULTS: Most specimens showed varying degrees of fibrosis. The degree of inflammation varied among the specimens, with half showing moderate or severe inflammatory changes. Fat deposition in hepatocytes was observed in almost all of the specimens, and severe fatty liver was noted in 20 (67%) of them. There was a mixture of two types of hepatocytes with macrovesicular or microvesicular fat droplets, and cholestasis was observed at a rate of 77%. Hemosiderin deposition, mostly mild and localized in periportal hepatocytes and macrophages in portal areas, was observed in 57% of the specimens. CONCLUSION: A combination of mixed macrovesicular and microvesicular fatty hepatocytes and the above-described findings, such as fatty liver, cholestasis, necroinflammatory reaction and iron deposition, are almost never observed in other liver diseases in infants and adults. We believe that NICCD is a disease with characteristic hepatopathological features.