Toshihiro Tajima

OTX2 loss of function mutation causes anophthalmia and combined pituitary hormone deficiency with a small anterior and ectopic posterior pituitary.

CONTEXTnOrthodenticle homeobox 2 (OTX2) is a transcription factor necessary for ocular and forebrain development. In humans, heterozygous mutations of OTX2 cause severe ocular malformations. However, whether mutations of OTX2 cause pituitary structural abnormalities or combined pituitary hormone deficiency (CPHD) has not been clarified.nnnOBJECTIVESnWe surveyed the functional consequences of a novel OTX2 mutation that was detected in a patient with anophthalmia and CPHD.nnnPATIENTnWe examined a Japanese patient with growth disturbance, anophthalamia, and severe developmental delay. He showed deficiencies in GH, TSH, LH, FSH, and ACTH. Brain magnetic resonance imaging revealed a small anterior pituitary gland, invisible stalk, ectopic posterior lobe, and Chiari malformation.nnnRESULTSnSequence analysis of OTX2 demonstrated a heterozygous two bases insertion [S136fsX178 (c.576-577insCT)] in exon 3. The mutant Otx2 protein localized to the nucleus, but did not activate the promoter of the HESX1 and POU1F1 gene, indicating a loss of function mutation. No dominant negative effect in the presence of wild-type Otx2 was observed.nnnCONCLUSIONnThis case indicates that the OTX2 mutation is a cause of CPHD. Further study of more patients with OTX2 defects is necessary to clarify the clinical phenotypes and endocrine defects caused by OTX2 mutations.

Elevated free thyroxine levels detected by a neonatal screening system.

In Sapporo city of Japan, neonatal screening for congenital hypothyroidism has used the measurement of free thyroxine (T4) and thyroid-stimulating hormone (TSH) in the filter-paper blood spot. This system has enabled us to identify hyperthyroxinemic diseases. Filter papers were collected from neonatal infants born at 4–6 d of age and neonates who showed elevated free T4 (>4.0 ng/dL, 4 SD above the mean) were studied. Between January 2000 and December 2006, 83,232 newborns were screened. Eleven infants demonstrated persistent hyperthyroxinemia. One patient with slightly elevated free T4 and normal TSH was diagnosed as having familial dysalbuminemic hyperthyroxinemia (FDH). The other two patients with elevated free T4 without suppressed TSH were considered as having resistance of thyroid hormone (RTH), and analysis of thyroid hormone receptor (TR) β gene confirmed the diagnosis. The remaining eight patients were diagnosed as having neonatal Graves disease (NGD). Seven of eight pregnant women were treated with antithyroid drug and thus only one unrecognized NGD during pregnancy was detected by screening. Our screening system enables for early awareness of RTH and FDH. Regarding Graves disease, the benefit of elevated free T4 screening is small, because most pregnant women with Graves disease were managed.

A novel mutation (V101A) of the LHX4 gene in a Japanese patient with combined pituitary hormone deficiency.

OBJECTIVEnLHX4, a LIM-homeodomain transcription factor, is required for development of the pituitary and nervous system. Several mutations of the LHX4 gene have been identified in patients with combined pituitary hormone deficiency (CPHD). The objective of the study was to clarify the molecular basis of a Japanese patient of CPHD with a small anterior pituitary and an ectopic posterior pituitary.nnnMETHODSnGenomic DNA was extracted from blood samples of the patient. Exons and exon-intron junctions of the LHX4 gene were amplified and sequenced. An expression vector of the mutant LHX4 protein was constructed and its function was analyzed in vitro.nnnRESULTSnA novel missense mutation (V101A) was identified. IN VITRO transfection studies demonstrated that V101A mutant LHX4 was unable to activate the POU1F1 and FSHbeta subunit gene promoter, indicating a loss of function mutation.nnnCONCLUSIONnOur results identify a novel loss of function mutation of the LHX4 gene in a Japanese patient with CPHD.

Congenital Hypothyroidism Caused by a PAX8 Gene Mutation Manifested as Sodium/Iodide Symporter Gene Defect.

Loss-of-function mutations of the PAX8 gene are considered to mainly cause congenital hypothyroidism (CH) due to thyroid hypoplasia. However, some patients with PAX8 mutation have demonstrated a normal-sized thyroid gland. Here we report a CH patient caused by a PAX8 mutation, which manifested as iodide transport defect (ITD). Hypothyroidism was detected by neonatal screening and L-thyroxine replacement was started immediately. Although 123I scintigraphy at 5 years of age showed that the thyroid gland was in the normal position and of small size, his iodide trapping was low. The ratio of the saliva/plasma radioactive iodide was low. He did not have goiter; however laboratory findings suggested that he had partial ITD. Gene analyses showed that the sodium/iodide symporter (NIS) gene was normal; instead, a mutation in the PAX8 gene causing R31H substitution was identified. The present report demonstrates that individuals with defective PAX8 can have partial ITD, and thus genetic analysis is useful for differential diagnosis.

Increased Na reabsorption via the Na–Cl cotransporter in autosomal recessive pseudohypoaldosteronism

BackgroundThe autosomal recessive form of pseudohypoaldosteronism type 1 (AR-PHA1) is caused by loss-of-function mutations in the epithelial sodium channel subunit genes and is characterized by a multisystemic and lifelong severe salt-wasting tendency. However, we observed a male AR-PHA1 patient who exhibited less frequent salt wasting with advancing age, despite the cessation of daily salt supplementation.ObjectiveTo elucidate the mechanism for the above phenomenon.MethodsWe evaluated the sodium-reabsorption ability of his distal nephrons (from the distal convoluted tubules to the collecting ducts) and compared it to that of a patient with the dominant form of PHA1 (AD-PHA1) carrying a heterozygous NR3C2 (mineralocorticoid receptor) gene mutation. In addition, immunoblotting of the thiazide-sensitive Na+–Cl− cotransporter (NCC) protein was conducted using urine samples from the AR- and AD-PHA1 patients.ResultsThe levels of sodium reabsorption that occurred via the distal nephrons were almost identical in the two PHA1 patients, despite their different molecular pathogeneses. Immunoblotting showed an increased urinary NCC protein level in the AR-PHA1 patient.ConclusionTaken together, increased sodium reabsorption via the upregulation of the expression of NCC might have been responsible, at least in part, for the clinical improvement seen in an AR-PHA1 patient.

Problems in diagnosing atypical Gitelman’s syndrome presenting with normomagnesaemia

Objectiveu2002 Gitelman’s syndrome, recognized as a variant of Bartter’s syndrome, is characterized by hypokalaemic metabolic alkalosis in combination with hypomagnesaemia and hypocalciuria. Overlapping biochemical features in Gitelman’s syndrome and Bartter’s syndrome has been observed. Here, we investigated the clinical, biochemical, and genetic characteristics of five, chronic, nonhypertensive and hypokalaemic Japanese patients.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis: unusual clinical associations and novel claudin16 mutation in an Egyptian family

BackgroundFamilial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive tubular disorder that eventually progresses to renal failure, depending upon the extent of nephrocalcinosis. Its basic pathogenesis is impaired tubular resorption of magnesium and calcium in the thick ascending limb of the loop of Henle (TAL) due to a genetic defect in paracellin-1 (a tight junction protein expressed in TAL). Mutations of the claudin16 gene (CLDN16), formerly called paracellin-1 gene (PCLN-1), have been linked to FHHNC.MethodsAn extended Egyptian family with more than one member affection by nephrocalcionsis was included and thoroughly investigated in the current study after giving informed consent. Thorough history was taken for polyuria, polydipsia and hypocalcemia symptoms, as well as clinical examination with stress on anthropometric measurements and radiological evaluation for kidneys and bones. Laboratory workup for the differential diagnosis of nephrocalcinosis was done: complete urinalysis, including urinary calcium excretion, urine pH and electrolytes, arterial blood gas (ABG), serum electrolytes (sodium, potassium, calcium, magnesium and phosphorous), renal function tests as well as parathyroid and gonadotropin-sex hormone assay. DNA extraction from peripheral blood leukocytes was done followed by amplification using primers previously described, purification and finally sequencing to analyze each exon of the CLDN16 gene.ResultsTwo sibs for a consanguineous couple were affected by nephrocalcinosis and showed persistent hypocalcemia, hypercalciuria, nephrocalcinosis with persistently alkaline urine and ocular manifestations in the form of congenital cataracts, high myopia and retinal abnormalities. The elder sib showed genitourinary abnormalities in the form of hypospadias and cryptorchidism. These two sibs had a homozygous two-base deletion in exon 1 of the CLDN16 gene (C. 233_234 del GG; Ins C), causing a frame shift mutation (Arg55xa0fs); however, their parents were heterozygote carriers for that mutation.ConclusionThe above-mentioned clinical data in the two affected sibs together with the family history of end-stage renal disease associated with nephrocalcinosis and high myopia suggested a diagnosis of FHHNC, which was confirmed for the first time in an Egyptian family by a novel mutation in exon 1 of the CLDN16 gene. Genitourinary associations with FHHNC have not yet been reported in the literature. Here, we will try to highlight the principles of mutation detection based on sequencing with the use of the online NCBI databases, statistics and other search tools.

Prenatal Diagnosis and Treatment of Steroid 21-Hydroxylase Deficiency

Steroid 21-hydroxylase deficiency (21-OHD) accounts for 90–95% of congenital adrenal hyperplasia (CAH) cases. It is classified into three distinct clinical phenotypes: the salt-wasting (SW), simple virilizing (SV) and nonclassical forms (NC). As girls with the SW and SV forms of 21-OHD are exposed to high systemic levels of adrenal androgens during fetal life, they show genital ambiguity. To ameliorate the degree of genital virilization, prenatal dexamethasone treatment has been performed for more than two decades, although mainly in the USA and Europe. This treatment has proven to be effective in preventing or reducing genital virilization. Some data also show that prenatal diagnosis and treatment are safe for the mother and fetus. However, prenatal treatment is still controversial for the following reasons. First, the risk of having an affected female fetus is only one in eight when both parents are known carriers of the autosomal recessive trait. Therefore, seven of eight fetuses will receive dexamethasone unnecessarily, and this raises ethical questions. Furthermore, maternal side effects such as excessive weight gain and hypertension have been observed. Finally, the long-term safety and outcome for dexamethasone-exposed children have not been established. In Japan, prenatal diagnosis and treatment has rarely been reported because of these reasons. Therefore, we must be cautious, and this treatment should be carried out in special centers with the approval of their ethical committees, that are capable of performing chorionic villus sampling (CVS) and subsequently determining the karyotype and genotype of 21-OHD.