A mutation of the ß-domain in POU1F1 causes pituitary deficiency due to dominant PIT-1ß expression.

BACKGROUND: POU1F1 encodes both PIT-1α, which plays pivotal roles in pituitary development and GH, PRL and TSHB expression, and the alternatively spliced isoform PIT-1ß, which contains an insertion of 26-amino acids (ß-domain) in the transactivation domain of PIT-1α due to the use of an alternative splice acceptor at the end of the first intron. PIT-1ß is expressed at much lower levels than PIT-1α and represses endogenous PIT-1α transcriptional activity. Although POU1F1 mutations lead to combined pituitary hormone deficiency (CPHD), no patients with ß-domain mutations have been reported. RESULTS: Here, we report that a three-generation family exhibited different degrees of CPHD, including growth hormone deficiency with intrafamilial variability of prolactin/TSH insufficiency and unexpected prolactinoma occurrence. The CPHD was due to a novel POU1F1 heterozygous variant (c.143-69T>G) in intron 1 of PIT-1α (RefSeq number NM_000306) or as c.152T>G (p.Ile51Ser) in exon 2 of PIT-1ß (NM_001122757). Gene splicing experiments showed that this mutation yielded the PIT-1ß transcript without other transcripts. The lymphocyte PIT-1ß mRNA expression was significantly higher in the patients with the heterozygous mutation than a control. A luciferase reporter assay revealed that the PIT-1ß-Ile51Ser mutant repressed PIT-1α and abolished transactivation capacity for the rat prolactin promoter in GH3 pituitary cells. CONCLUSIONS: We describe, for the first time, that the PIT-1ß mutation can cause CPHD through a novel genetic mechanism, such as PIT-1ß overexpression, and that POU1F1 mutation might be associated with a prolactinoma. Analysis of new patients and long-term follow-up are needed to clarify the characteristics of PIT-1ß mutations.

Increase in doses of levothyroxine at the age of 3 years and above is useful for distinguishing transient and permanent congenital hypothyroidism.

There are no recommended diagnostic criteria for transient congenital hypothyroidism (CH) during early childhood. In this study, we aimed to identify the factors that distinguish permanent (P)- and transient (T)-CH. We retrospectively analyzed the clinical, biochemical, and imaging data of 42 children with a definitive diagnosis of P- or T-CH by re-evaluation tests at our institution from November 1986 to October 2019. Patients who continued levothyroxine (L-T4) treatment after the re-evaluation tests were classified as group P (n = 19), while patients who were diagnosed with T-CH and discontinued L-T4 treatment were classified as group T (n = 23). Initial testing performed during infancy showed that the mean serum TSH and free T4 (FT4) levels did not differ significantly between groups P and T. None of the patients in group T required an increased dosage of L-T4 at the age of 3 yr and above while 85% of the patients in group P required increased dosages of L-T4. Hence, T-CH was suspected in patients who did not require an increase in L-T4 dosage at the age of 3 yr and above.

A 34-year-old Japanese patient exhibiting NBAS deficiency with a novel mutation and extended phenotypic variation.

Biallelic neuroblastoma amplified sequence (NBAS) gene mutations have recently been identified to cause a reduction in its protein expression and a broad phenotypic spectrum, from isolated short stature, optic nerve atrophy, and Pelger-Huët anomaly (SOPH) syndrome or infantile liver failure syndrome 2 to a combined, multi-systemic disease including skeletal dysplasia and immunological and neurological abnormalities. Herein, we report a 34-year-old patient with a range of phenotypes for NBAS deficiency due to compound heterozygous variants; one is a SOPH-specific variant, p.Arg1914His, and the other is a novel splice site variant, c.6433-2A>G. The patient experienced recurrent acute liver failure until early childhood. Hypogammaglobulinemia, a decrease in natural killer cells, and optic nerve atrophy were evident from infancy to childhood. In adulthood, the patient exhibited novel phenotypic features such as hepatic cirrhosis complicated by portal hypertension and autoimmune hemolytic anemia. The patient also suffered from childhood-onset insulin-requiring diabetes with progressive beta cell dysfunction. The patient had severe short stature and exhibited dysmorphic features compatible with SOPH, intellectual disability, and epilepsy. NBAS protein expression in the patient's fibroblasts was severely low. RNA expression analysis for the c.6433-2A>G variant showed that this variant activated two cryptic splice sites in intron 49 and exon 50, for which the predicted consequences at the protein level were an in-frame deletion/insertion, p.(Ile2199_Asn2202delins16), and a premature termination codon, p.(Ile2199Tyrfs*17), respectively. These findings indicate that NBAS deficiency is a multi-systemic progressive disease. The results of this study extend the spectrum of clinical and genetic findings related to NBAS deficiency.

Glycemic control indicator levels at diagnosis of neonatal diabetes mellitus: Comparison with other types of insulin-dependent diabetes mellitus.

BACKGROUND: Neonatal diabetes mellitus (NDM) is a monogenic insulin-dependent diabetes that develops within 6 months of age. The progression of hyperglycemia until diagnosis is unknown. Glycemic control indicators at diagnosis are useful to estimate the extent and duration of hyperglycemia. We recently established that age-adjusted glycated albumin (GA) is a useful indicator of glycemic control, regardless of age. OBJECTIVE: To compare the levels of various glycemic control indicators at diagnosis between NDM and other types of insulin-dependent diabetes mellitus. PATIENTS AND METHODS: We included 8 patients with NDM, 8 with fulminant type 1 diabetes (FT1D), and 24 with acute-onset autoimmune type 1 diabetes (T1AD). Plasma glucose, glycated hemoglobin (HbA1c), GA, and age-adjusted GA (calculated as previously reported) were measured and compared. RESULTS: There were no significant differences in the plasma glucose levels of the group of patients with NDM and those with FT1D or T1AD. HbA1c and GA levels in the NDM group were not significantly different from those in the FT1D group, and both indicators were lower than those in the T1AD group. Age-adjusted GA levels in the NDM group did not differ significantly from those in the T1AD group, but were higher than those in the FT1D group. CONCLUSIONS: These findings suggest that the time-course of plasma glucose elevation in NDM until diagnosis is similar to that in T1AD. In addition, the high age-adjusted GA value at diagnosis of NDM indicates that this test is useful for assessing chronic hyperglycemia in NDM.

Establishment of monoclonal antibodies against cell surface domains of ASCT2/SLC1A5 and their inhibition of glutamine-dependent tumor cell growth.

Human alanine-serine-cysteine transporter 2 (ASCT2; SLC1A5) is a major transporter of the amino acid glutamine that is known to be overexpressed in certain malignant tumors. In this study, we generated specific monoclonal antibodies (MAbs) against ASCT2 by establishing an ASCT2-expressing Chinese hamster ovary cell line that was used to immunize mice and rats. The MAbs KM4008, KM4012, and KM4018 against ASCT2 were isolated through a cell-based screen; these specifically bound to ASCT2-positive cells, as determined by flow cytometry and immunoprecipitation. In addition, the antibodies suppressed glutamine-dependent growth of WiDr colorectal cancer cells. These results provide evidence supporting the use of MAbs against ASCT2 as an effective therapeutic strategy for cancer treatment.

A Novel Mutation in the type Iα Regulatory Subunit of Protein Kinase A (PRKAR1A) in a Cushing's Syndrome Patient with Primary Pigmented Nodular Adrenocortical Disease.

A 40-year-old man presented with Cushing's syndrome due to bilateral adrenal hyperplasia with multiple nodules. Computed tomography scan results were atypical demonstrating an enlargement of the bilateral adrenal glands harboring multiple small nodules, but the lesion was clinically diagnosed to be primary pigmented nodular adrenocortical disease (PPNAD) based on both endocrinological test results and his family history. We performed bilateral adrenalectomy and confirmed the diagnosis histologically. An analysis of the patient and his mother's genomic DNA identified a novel mutation in the type Iα regulatory subunit of protein kinase A (PRKAR1A) gene; p.E17X (c.49G>T). This confirmed the diagnosis of PPNAD which is associated with Carney Complex.

Age-Adjusted Glycated Albumin at Diagnosis is more Correlated with the Product of Age and Plasma Glucose than Plasma Glucose Alone in Patients with Neonatal Diabetes Mellitus.

BACKGROUND: We previously showed that glycated albumin (GA) is a useful glycemic control indicator in patients with neonatal diabetes mellitus (NDM), and that age-adjusted GA (Aa-GA) can reflect more accurately glycemic control status. Here, we investigated whether the age at diagnosis influences Aa-GA at diagnosis of NDM. METHODS: Eight patients with NDM whose GA was measured at diagnosis (age at diagnosis: 39 ± 18 days; GA: 31.3 ± 7.6%; Aa-GA: 47.1 ± 10.3%; plasma glucose: 525 ± 194 mg/dl) were included. Aa-GA was calculated as follows: Aa-GA = GA × 14.0/[1.77 × log-age (days) + 6.65]. Correlations of GA or Aa-GA at diagnosis with its logarithmically transformed age in days (log-age), plasma glucose, and their product were investigated. RESULTS: GA at diagnosis was not significantly correlated with log-age or plasma glucose. On the other hand, Aa-GA at diagnosis was significantly positively correlated with plasma glucose (R = 0.75, P = 0.031) and was more strongly positively correlated with the product of plasma glucose and log-age (R = 0.82, P = 0.012) although it was not correlated with log-age. CONCLUSION: Aa-GA at diagnosis is influenced by both age in days and plasma glucose. This finding is likely to show the aspect that age in days is almost equal to diabetes duration because glycemic control indicators including GA reflect the weighted mean of plasma glucose.

Age-adjusted glycated albumin accurately reflects blood glucose in patients with neonatal diabetes mellitus: comparison with calculated glycated albumin determined by past blood glucose concentrations.

BACKGROUND: Glycated albumin is a useful glycaemic control indicator for neonatal diabetes mellitus. However, glycated albumin concentrations in infants are lower than those in adults and increase in an age-dependent manner. Based on our investigation of non-diabetic subjects, we proposed the possibility that the reference range for adults may be used regardless of age, provided that age-adjusted glycated albumin is employed. In the present study, we evaluate the usefulness of age-adjusted glycated albumin in neonatal diabetes mellitus patients. METHODS: Six neonatal diabetes mellitus patients (four patients with permanent neonatal diabetes mellitus and two patients with transient neonatal diabetes mellitus) were included. Measured glycated albumin or age-adjusted glycated albumin was compared to calculated glycated albumin, which was determined using calculation formulae we had reported based on past blood glucose over the 50 days before measurement of glycated albumin. RESULTS: Measured glycated albumin was significantly lower than calculated glycated albumin (20.5 ± 4.9% versus 28.2 ± 6.1%; p < 0.0001), whereas age-adjusted glycated albumin was equivalent to calculated glycated albumin, showing no significant difference (27.5 ± 6.8% versus 28.2 ± 6.1%). Measured glycated albumin concentrations in patients with transient neonatal diabetes mellitus in remission were lower than the reference range for adults, whereas age-adjusted glycated albumin concentrations were within the reference range for adults. CONCLUSION: We demonstrated that age-adjusted glycated albumin concentrations were consistent with calculated glycated albumin. Age-adjusted glycated albumin is therefore a useful glycaemic control indicator for neonatal diabetes mellitus patients.

Generation of specific monoclonal antibodies against the extracellular loops of human claudin-3 by immunizing mice with target-expressing cells.

Human claudin-3 (CLDN3) is a tetraspanin transmembrane protein of tight junction structures and is known to be over-expressed in some malignant tumors. Although a specific monoclonal antibody (MAb) against the extracellular domains of CLDN3 would be a valuable tool, generation of such MAbs has been regarded as difficult using traditional hybridoma techniques, because of the conserved sequence homology of CLDN3s among various species. In addition, high sequence similarity is shared among claudin family members, and potential cross-reactivity of MAb should be evaluated carefully. To overcome these difficulties, we generated CLDN3-expressing Chinese hamster ovary and Sf9 cells to use an immunogens and performed cell-based screening to eliminate cross-reactive antibodies. As a result, we generated MAbs that recognized the extracellular loops of CLDN3 but not those of CLDN4, 5, 6, or 9. Further in vitro studies suggested that the isolated MAbs possessed the desired binding properties for the detection or targeting of CLDN3.

Glycemic control and motor development in a patient with intermediate DEND.

The most common cause of neonatal diabetes, KCNJ11 gene mutation, can manifest as a neurological disorder. The most severe form consists of a constellation of developmental delay, epilepsy, and neonatal diabetes (DEND). Intermediate DEND (iDEND) refers to a milder presentation without epilepsy. We present a child with iDEND, for whom insulin injections were replaced with glibenclamide therapy at 17 months of age because of poor glycemic control and delayed motor development. Three months after initiation of glibenclamide, HbA1c decreased from 10.2% to 5.6%. Continuous glucose monitoring indicated that blood glucose fluctuations were suppressed while on glibenclamide. Furthermore, after initiating glibenclamide therapy, the developmental quotient (DQ) for motor ability markedly improved from 60 to 91, whereas the DQ for language and adoptive ability remained as they had been before the sulfonylurea treatment. Sulfonylurea treatment improved glycemic control and motor development in the present patient.

HbA1c can be a useful glycemic control marker for patients with neonatal diabetes mellitus older than 20 weeks of age.

BACKGROUND: The accuracy of most HbA1c analysis methods is affected by the presence of increased fetal hemoglobin (HbF). The objective of this study was to investigate the age at which HbA1c measurements become useful for monitoring glycemic control in patients with neonatal diabetes mellitus (NDM). METHODS: We retrospectively analyzed the data of 5 NDM patients diagnosed at 38±20 days of age, who each had several available HbA1c measurements during the first year of life, with a control group of HbA1c values over the course of 1 year for 13 patients with type 1 diabetes mellitus (T1DM). Mean blood glucose (MBG) levels derived from premeal or premeal plus bedtime blood glucose measurements prior to HbA1c measurements were compared to HbA1c values. RESULTS: The NDM patients' age at which the difference in the HbA1c/MBG ratios became not significant between the NDM patients and the T1DM patients was 21 weeks of age and over. Even after the HbA1c was adjusted for HbF, this ratio was significantly lower in the NDM patients at <21 weeks of age than in the T1DM patients. CONCLUSIONS: HbA1c can be a useful glycemic control marker for NDM patients >20 weeks of age.

Age-adjusted glycated albumin: a more robust parameter to establish glycaemic control in neonatal diabetes mellitus.

BACKGROUND: We previously reported that glycated albumin (GA) levels increased in an age-dependent manner in infancy. In order to determine whether this phenomenon is true from infancy to adulthood, we investigated the GA levels in non-diabetic subjects of a wide range of age. METHODS: GA levels of 376 non-diabetic subjects [average age, 31.8 ± 23.8 years (4 days-78 years)] were determined. A relationship between GA and logarithmically transformed age [log(age)] was analysed. RESULTS: GA levels were significantly positively correlated with log(age) [R = 0.865, P < 0.0001, GA = 1.77 × log(day) + 6.55]. Based on a regression line, we established the formula for adjusting GA levels according to age. CONCLUSION: We showed that GA increases with age from infancy to adulthood and that normal GA levels are demonstrated as a simple regression formula with log(age). This formula allowing us to use the adult reference range has the potential for treatment monitoring of diabetic patients regardless of age.

A case of pancreatic agenesis and congenital heart defects with a novel GATA6 nonsense mutation: evidence of haploinsufficiency due to nonsense-mediated mRNA decay.

Recently, GATA6 heterozygous loss-of-function mutations were reported to cause pancreatic agenesis and congenital heart defects (PACHD [OMIM:600001]). However, the molecular mechanisms resulting from premature termination codons have not been examined in this disorder. The objective of this study was to perform a genetic analysis of a patient with PACHD. A female patient presented with ventricular septal defect, patent ductus arteriosus, and congenital diaphragmatic hernia at birth. Permanent neonatal diabetes mellitus and pancreatic exocrine deficiency due to pancreatic agenesis was diagnosed at 1 month of age. PCR-direct sequencing of GATA6 revealed that the patient is heterozygous for a novel de novo nonsense mutation of c.1477C>T, p. Arg493X in exon 5. RT-PCR direct sequencing of the RT-PCR products of total RNA from peripheral blood of the patient for the region encompassing exons 4-6 revealed only the wild-type allele. This finding provides the evidence for the occurrence of nonsense-mediated mRNA decay (NMD) in the p.Arg493X mutation. Quantitative RT-PCR analysis revealed that the expression of GATA6 transcript in the patient was less than half compared with normal control samples. This is the first evidence that GATA6 haploinsufficiency is caused by NMD in vivo, and we conclude that GATA6 haploinsufficiency causes not only PACHD but may affect other organs derived from the endoderm. Further screenings of GATA6 mutations in patients with various forms of diabetes and/or congenital heart disease with other visceral malformation may reveal the impact of GATA6 mutations on diabetes and congenital malformation.

A Case of Vitamin D Deficiency without Elevation of Serum Alkaline Phosphatase in a Carrier of Hypophosphatasia.

Elevated serum alkaline phosphatase (ALP) is a screening marker for the diagnosis of vitamin D deficiency, which may fail to be diagnosed if serum ALP is not elevated. Here, we describe a case of vitamin D deficiency without elevation of serum ALP. A 1-year-old Japanese girl was referred to our hospital for the evaluation of genu varum. Her serum intact PTH level was elevated, while her serum ALP level was normal. Furthermore, her serum 25-hydroxyvitamin D level was reduced, and her urine phosphoethanolamine (PEA) level was mildly elevated. ALPL gene analysis revealed she was a heterozygous carrier of hypophosphatasia (c.1559delT). Serum intact PTH and urine PEA evaluations were helpful for diagnosing vitamin D deficiency and hypophosphatasia carrier status, respectively. Therefore, the possibility of vitamin D deficiency without elevation of serum ALP should be considered.

Evaluation of glycated hemoglobin and fetal hemoglobin-adjusted HbA1c measurements in infants.

BACKGROUND: As the presence of fetal hemoglobin (HbF) affects the accuracy of hemoglobin A1c (HbA1c) analysis methods, HbA1c measurement may not be a good indicator for patients with neonatal diabetes mellitus, whereas glycated albumin (GA) may be a good indicator. OBJECTIVE: To investigate whether total glycated hemoglobin (GHb) or HbF-adjusted HbA1c (adj-HbA1c) can act as a glycemic control marker in infants. SUBJECTS AND METHODS: Plasma glucose (PG), GA, HbF, GHb measured using the affinity method, and HbA1c measured using the latex-immunoturbidimetry (LA) or the high-performance liquid chromatography (HPLC) methods were determined in 26 full-term newborn infants aged 4-234 d. Adj-HbA1c was calculated as HbA1c/(total Hb - HbF). RESULTS: GHb, adj-HbA1c measured using the LA and the HPLC methods were 4.8 ± 0.5%, 4.5 ± 0.5%, and 4.7 ± 0.6%, respectively. GA was most positively correlated with PG (r = 0.696, p < 0.0001). GHb was positively correlated with both PG (r = 0.479, p = 0.013) and GA (r = 0.727, p < 0.0001). Adj-HbA1c measured using the LA method was positively correlated with GA (r = 0.465, p = 0.017), but not PG (r = 0.304, p = 0.132). Adj-HbA1c measured using the HPLC method was correlated with neither PG (r = -0.077, p = 0.710) nor GA (r = 0.360, p = 0.071). CONCLUSIONS: GHb measured using the affinity method may be a useful glycemic control marker in infants. Although adj-HbA1c measured using the LA method was correlated with GA, it may not be a practical measure because it was not correlated with PG and determining HbF levels using HPLC method can be troublesome. Adj-HbA1c measured using the HPLC method should not be used as a glycemic marker in infants.

Glycated albumin is lower in infants than in adults and correlated with both age and serum albumin.

BACKGROUND: Glycated albumin (GA) reflects glycemic control in patients with neonatal diabetes mellitus (NDM). However, GA in NDM patients is apparently low in relation to glycemia. OBJECTIVE: To establish the reference intervals for GA in healthy infants. SUBJECTS AND METHODS: Fifty-eight healthy, full-term newborn infants were used to define the GA reference values and to investigate its relationship to plasma glucose (PG) and serum albumin. The infants were categorized into three groups according to age: group A, 5 (4-6) median (range) d: n = 18; group B, 33 (30-38) d: n = 19; and group C, 181 (50-352) d: n = 21. We also studied 212 non-diabetic adults [group D, 53 (28-78) yr old] and the 5 NDM patients previously reported for GA comparisons. RESULTS: In the infants, GA was strongly positively correlated with logarithmic transformation of age [log (age)] (p = 0.831, p < 0.0001). The GA in groups A, B, C, and D were 7.3 ± 1.0%, 8.6 ± 1.1%, 10.9 ± 0.8%, and 14.0 ± 1.1%, respectively. The GA was more strongly positively correlated with serum albumin (r = 0.768, p < 0.0001) than with PG (r = 0.596, p < 0.0001). When GA levels were compared with the age-dependent reference values, GA in the transient NDM patient was normalized although GA in the four permanent NDM patients decreased but remained high after insulin therapy. CONCLUSIONS: This study showed that the reference range for GA in infants is lower than that of adults and increases with age, with which we confirmed that GA in the NDM patients reflected the clinical course. Consequently, GA in NDM patients should be compared with the age-based reference values to assess the accurate glycemic status.