Tohru Yorifuji

Craniotabes in Normal Newborns: The Earliest Sign of Subclinical Vitamin D Deficiency

CONTEXT Craniotabes in otherwise normal neonates has been regarded as physiological and left untreated. OBJECTIVE Our objective was to investigate the role of vitamin D deficiency in the development of craniotabes in normal neonates. DESIGN AND SETTING Newborn screening of craniotabes was conducted at the single largest obstetrical facility in Kyoto, Japan. Follow-up study at 1 month was conducted at Kyoto University Hospital. SUBJECTS A total of 1120 consecutive normal Japanese neonates born in May, 2006, through April, 2007, were included in the study. MAIN OUTCOME MEASURES The incidence of craniotabes was scored each month. Neonates with craniotabes were followed up at 1 month with measurements of serum calcium, phosphorus, alkaline phosphatase (ALP), intact PTH, 25-OH vitamin D (25-OHD), urinary calcium, phosphorus, creatinine, and hand x-rays. RESULTS Craniotabes was present in 246 (22.0%) neonates, and the incidence had obvious seasonal variations, highest in April-May and lowest in November. At 1 month, infants with craniotabes had significantly higher serum ALP compared with normal neonates; 6.9% of them had elevated intact PTH over 60 pg/ml, and 37.3% had 25-OHD less than 10 ng/ml. When separately analyzed according to the method of feeding, 56.9% of breast-fed infants showed 25-OHD less than 10 ng/ml, whereas none of formula/mixed-fed infants did, and breast-fed infants had significantly higher serum PTH and ALP compared with formula/mixed-fed infants. SUMMARY These results suggest that craniotabes in normal neonates is associated with vitamin D deficiency in utero, and the deficiency persists at 1 month in many of them, especially when breast-fed.

Unfavorable effects of growth hormone therapy on the final height of boys with short stature not caused by growth hormone deficiency

A group of 18 boys with non-growth hormone (GH)-deficient short stature without GH therapy (group A) and another group of 9 boys with non-GH-deficient short stature with GH therapy in doses of 0.5 IU (0.17 mg)/kg per week administered 5 to 6 times weekly (group B) were observed until they reached their final height. The mean duration of GH therapy was 4.2 years (range 3.2 to 5.0 years). These two groups were matched with respect to their standard deviation score (SDS) for bone age at the start of observation. Mean +/- SD of the final height for group A and group B was 162.0 +/- 5.4 cm and 154.2 +/- 4.2 cm, respectively. During the prepubertal period, height SDS for bone age of these two groups was not affected by GH therapy. During the pubertal period, however, height SDS for bone age remained constant for group A but decreased gradually for group B. Our observation indicates that for boys with non-GH-deficient short stature GH therapy does not improve height SDS for bone age during the prepubertal period, and in fact reduces it during the pubertal period, possibly resulting in a shorter final height than might have been attained naturally.

Formation of Advanced Glycosylation End Products and Oxidative Stress in Young Patients with Type 1 Diabetes

Increased production of advanced glycosylation end products (AGEs) and augmented oxidative stress may contribute to vascular complications in diabetes. Little is known about the formation and accumulation of AGEs in young patients with type 1 diabetes. The aim of the present study was to investigate whether AGE production and oxidative stress are augmented in young patients with type 1 diabetes at early clinical stages of the disease. Urine samples of 38 patients with type 1 diabetes [mean age (±SD), 12.8 ± 4.5 y; diabetes duration, 5.7 ± 4.3 y; HbA1c, 8.0 ± 1.6%; urinary albumin excretion, 12.6 ± 14.4 mg/g creatinine (Cr)] and those of 60 age-matched healthy control subjects were assayed for AGEs, pentosidine and pyrraline, and markers of oxidative stress, 8-hydroxy-2′-deoxyguanosine (8-OHdG) and acrolein-lysine. Of these four markers, urinary concentrations of pentosidine, 8-OHdG, and acrolein-lysine were significantly higher in the patients with diabetes than in the healthy control subjects. For the patient group, pentosidine correlated significantly with 8-OHdG and acrolein-lysine, and pyrraline correlated significantly with acrolein-lysine. Urinary pentosidine, 8-OHdG, and acrolein-lysine but not pyrraline correlated significantly with urinary albumin excretion. Patients with microalbuminuria (≥15 mg/g Cr) showed significantly higher levels of all four markers than did normoalbuminuric patients and control subjects. The present study indicates that accumulation of AGEs, whose formation is closely linked to oxidative stress, and resultant endothelial dysfunction may start early in the course of type 1 diabetes. This means that the risk of vascular complications may be present at an early age and that the best possible glycemic control should be emphasized from the diagnosis of diabetes.

Skewed X inactivation in manifesting carriers of Duchenne muscular dystrophy

We studied X inactivation patterns in manifesting carriers of familial and sporadic Duchenne muscular dystrophy (DMD) or unaffected carriers of DMD by analysis of the methylation of Hpall sites in the first exon of the human androgen‐receptor gene (HUMARA) from peripheral blood samples. Three of the four manifesting carriers, four of the five asymptomatic carriers, and 31 of the 32 female controls were heterozygous for the CAG repeat of HUMARA. All manifesting carriers showed skewed X inactivation, while all unaffected carriers showed almost symmetrical inactivation. One family studied over three generations is noteworthy because it includes two mother/daughter pairs, one an affected pair with skewed X inactivation, and the other a phenotypically normal carrier pair with random X inactivation. On the other hand, the extent of X inactivation for each X chromosome in 31 female controls was widely distributed. These data suggest that in carriers of DMD, both affected and unaffected, it is valuable to analyze the pattern of skewed X inactivation because it provides important prognostic information. Carriers of DMD with skewed X inactivation might show slowly progressive myopathy with advancing age.

X-inactivation pattern in the liver of a manifesting female with ornithine transcarbamylase (OTC) deficiency

Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle disorder. It is X‐linked and hemizygous new‐born males usually suffer fatal hyperammonemia. In contrast, carrier females manifest variable phenotypes, ranging from asymptomatic carriers to those with severe hyperammonemia. In order to understand the correlation between X‐inactivation status and the clinical phenotype of carrier females with this disorder, we analyzed the X‐inactivation pattern of peripheral blood leukocytes in a family consisting of a clinically normal mother and two daughters with severe manifestation. In addition, we obtained tissue samples from various parts of the liver of one of these daughters and analyzed X‐inactivation patterns and the residual OTC activities. The X‐inactivation of peripheral blood leukocytes was nearly random in these carrier females and showed no correlation with the disease phenotype. However, the X‐inactivation of the liver was much more skewed and correlated well with the OTC activity of all samples. Interestingly, the degree of X‐inactivation varied considerably, even within the same liver.

Mucolipidosis II and III alpha/beta: mutation analysis of 40 Japanese patients showed genotype–phenotype correlation

Mucolipidosis (ML) II alpha/beta and III alpha/beta are autosomal recessive diseases caused by a deficiency of α and/or β subunits of the enzyme N-acetylglucosamine-1-phosphotransferase, which is encoded by the GNPTAB gene. We analyzed the GNPTAB gene in 25 ML II and 15 ML III Japanese patients. In most ML II patients, the clinical conditions ‘stand alone’, ‘walk without support’ and ‘speak single words’ were impaired; however, the frequency of ‘heart murmur’, ‘inguinal hernia’ and ‘hepatomegaly and/or splenomegaly’ did not differ between ML II and III patients. We detected mutations in GNPTAB in 73 of 80 alleles. Fourteen new mutations were c.914_915insA, c.2089_2090insC, c.2427delC, c.2544delA, c.2693delA, c.3310delG, c.3388_3389insC+c.3392C>T, c.3428_3429insA, c.3741_3744delAGAA, p.R334L, p.F374L, p.H956Y, p.N1153S and duplication of exon 2. Previously reported mutations were p.Q104X, p.W894X, p.R1189X and c.2715+1G>A causing skipping of exon 13. Homozygotes or compound heterozygotes of nonsense and frameshift mutations contributed to the severe phenotype. p.F374L, p.N1153S and splicing mutations contributed to the attenuated phenotype, although coupled with nonsense mutation. These results show the effective molecular diagnosis of ML II and III and also provide phenotypic prediction. This is the first and comprehensive report of molecular analysis for ML patients of Japanese origin.

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.

Acquired growth hormone deficiency due to pituitary stalk transection after head trauma in childhood

Two patients are reported with growth hormone deficiency due to head trauma in childhood. Although their injuries were outwardly only slight and there was no loss of consciousness and no subsequent neurological deficits, they exhibited gradual growth retardation from the time of the trauma. Provocative endocrinological tests showed growth hormone deficiency and MRI showed transection of the pituitary stalk. These findings suggest that ordinary head trauma, as well as perinatal insult and congenital abnormalities, could be a cause of growth hormone deficiency.

Comprehensive molecular analysis of Japanese patients with pediatric-onset MODY-type diabetes mellitus

Yorifuji T, Fujimaru R, Hosokawa Y, Tamagawa N, Shiozaki M, Aizu K, Jinno K, Maruo Y, Nagasaka H, Tajima T, Kobayashi K, Urakami T. Comprehensive molecular analysis of Japanese patients with pediatric‐onset MODY‐type diabetes mellitus.

Allograft steatohepatitis in progressive familial intrahepatic cholestasis type 1 after living donor liver transplantation

We studied histological features and long‐term outcomes in patients with progressive familial intrahepatic cholestasis type 1 (PFIC1) after liver transplantation (LT). Histological findings were correlated with the post‐LT course and treatment in 11 recipients with PFIC1. Ages at LT varied from 1 to 18 years (median, 4 years). Macrovesicular steatosis was observed in 8 patients at a median of 60 days post‐LT (range, 21‐191 days). Severe steatosis progressed to steatohepatitis in 7 patients at a median of 161 days (range, 116‐932 days). The patients were followed up for a median of 7.3 years (range, 2.3‐16.1 years). Six showed bridging fibrosis, with 2 progressing to cirrhosis. One patient with cirrhosis died because of the rupture of a splenic artery aneurysm 13.6 years post‐LT. Post‐LT refractory diarrhea was present in all 8 having steatosis. Three without post‐LT diarrhea showed no allograft steatosis. Bile adsorptive resin therapy reduced the diarrhea and steatosis. Patients with posttransplant steatosis typically had more severe mutations of the ATPase class I type 8B member 1 (ATP8B1) gene and were more likely to have systemic complications such as pancreatitis. In conclusion, allograft steatosis was present in patients with PFIC1, progressing to steatohepatitis and cirrhosis. Because expression of the familial intrahepatic cholestasis 1 gene occurs in several organs, including the small intestine, pancreas, and liver, and it is involved in enterohepatic bile acid circulation, post‐LT steatosis may be due to a malfunction of the ATP8B1 product. Liver Transpl 15:610–618, 2009.