Ayako Chida

Outcomes of childhood pulmonary arterial hypertension in BMPR2 and ALK1 mutation carriers

Mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene and the activin receptor-like kinase 1 (ALK1) gene have been reported in heritable pulmonary arterial hypertension (HPAH) and idiopathic pulmonary arterial hypertension (IPAH). However, the relation between clinical characteristics and each gene mutation in IPAH and HPAH is still unclear, especially in childhood. The aim of this study was to determine, in a retrospective study, the influence and clinical outcomes of gene mutations in childhood IPAH and HPAH. Fifty-four patients with IPAH or HPAH whose onset of disease was at <16 years of age were included. Functional characteristics, hemodynamic parameters, and clinical outcomes were compared in BMPR2 and ALK1 mutation carriers and noncarriers. Overall 5-year survival for all patients was 76%. Eighteen BMPR2 mutation carriers and 7 ALK1 mutation carriers were detected in the 54 patients with childhood IPAH or HPAH. Five-year survival was lower in BMPR2 mutation carriers than mutation noncarriers (55% vs 90%, hazard ratio 12.54, p = 0.0003). ALK1 mutation carriers also had a tendency to have worse outcome than mutation noncarriers (5-year survival rate 64%, hazard ratio 5.14, p = 0.1205). In conclusion, patients with childhood IPAH or HPAH with BMPR2 mutation have the poorest clinical outcomes. ALK1 mutation carriers tended to have worse outcomes than mutation noncarriers. It is important to consider aggressive treatment for BMPR2 or ALK1 mutation carriers.

Mutations of NOTCH3 in childhood pulmonary arterial hypertension

Mutations of BMPR2 and other TGF‐β superfamily genes have been reported in pulmonary arterial hypertension (PAH). However, 60–90% of idiopathic PAH cases have no mutations in these genes. Recently, the expression of NOTCH3 was shown to be increased in the pulmonary artery smooth muscle cells of PAH patients. We sought to investigate NOTCH3 and its target genes in PAH patients and clarify the role of NOTCH3 signaling. We screened for mutations in NOTCH3, HES1, and HES5 in 41 PAH patients who had no mutations in BMPR2, ALK1, endoglin, SMAD1/4/8, BMPR1B, or Caveolin‐1. Two novel missense mutations (c.2519 G>A p.G840E, c.2698 A>C p.T900P) in NOTCH3 were identified in two PAH patients. We performed functional analysis using stable cell lines expressing either wild‐type or mutant NOTCH3. The protein‐folding chaperone GRP78/BiP was colocalized with wild‐type NOTCH3 in the endoplasmic reticulum, whereas the majority of GRP78/BiP was translocated into the nuclei of cells expressing mutant NOTCH3. Cell proliferation and viability were higher for cells expressing mutant NOTCH3 than for those expressing wild‐type NOTCH3. We identified novel NOTCH3 mutations in PAH patients and revealed that these mutations were involved in cell proliferation and viability. NOTCH3 mutants induced an impairment in NOTCH3‐HES5 signaling. The results may contribute to the elucidation of PAH pathogenesis.

Severe human herpesvirus 6-associated encephalopathy in three children: analysis of cytokine profiles and the carnitine palmitoyltransferase 2 gene.

Three children developed severe encephalopathy associated with human herpesvirus 6 infection. Magnetic resonance imaging of the brain showed either basal ganglia involvement or diffusion abnormalities in the cerebral white matter. Coagulopathy with hypercytokinemia was observed in 2 patients. One demonstrated thermolabile variation in carnitine palmitoyltransferase 2. These results suggest a heterogeneous pathogenic mechanism in encephalopathy associated with human herpesvirus 6 infection.

A non‐obese boy with Prader‐Willi syndrome shows cardiopulmonary impairment due to severe kyphoscoliosis

Prader-Willi syndrome (PWS, OMIM #176270) is a genetic disorder characterized by hypotonia, obesity, characteristic facial appearance, hypogonadism, short stature, and behavioral abnormalities. It results from deletion or disruption of one or more genes on the proximal long arm of the paternally derived chromosome 15, or from maternal uniparental disomy 15. Musculoskeletal abnormalities are common in these individuals. Scoliosis is very common in PWS being variously reported in 62–86% of patients [Holm and Laurnen, 1981; Laurance et al., 1981]. Here, we describe a non-obese boy with PWS who also presented with cardiopulmonary impairment due to severe kyphoscoliosis. This secondary manifestation was described by Guilleminault et al. [1981]. The male patient was born to healthy and nonconsanguineous parents at 41 weeks of gestation, with a birth weight of 2,430 g ( 1.9 SD) and length of 47.0 cm ( 1.4 SD). Other family members were healthy. He showed hypotonia with poor suck in infancy. He held his head at age 9 months, sat at 1 year, and walked at 41⁄2years. He had a normal male karyotype (46,XY) and therewasno SNRPN signal on FISH. Kyphoscoliosis that was recognized at 1 year of age had progressively deteriorated (Fig. 1A,B). He had received growth hormone (GH) therapy (0.175 mg/kg/week) from age 10 years. He had a restrictive deficit on spirometry at age 1011=12 years: the median forced expiratory volume in 1 sec (FEV1) and forced vital capacity (FVC) were 1.03 L and 1.15 L, respectively. Although he had no respiratory symptoms, he underwent combined anterior fusion with posterior corrective fusion surgery using spinal instrumentation for kyphoscoliosis at age 11 years. However, the instrumentation had to be removed due to deep postoperative infection, which did not resolve until age 116=12 years. His weight management had been controlled well without any obesity from birth. At age 1510=12 years, he was admitted because of a 1-month history of shortness of breath and edema of the legs. On admission, he measured 133.0 cm and weighed 41.2 kg. Under normal conditions, his weight was 33.0 kg and his calculated body mass index (BMI) was 18.7 kg/m. He had a characteristic facial appearance, narrow hands with a straight ulnar border, and chilblain scars on the legs. Physical examination revealed tachypnea, tachycardia, cyanosis, a systolic heart murmur and gallop rhythm, distended abdomen, and edema of the whole body, especially of the legs. Neurological examination showed normal deep tendon reflexes and mild hypotonia. Laboratory tests showed respiratory acidosis, hypercapnia, and heart failure: pH, 7.285; pCO2, 70.6 mmHg in room air; hANP, 557 pg/ml; and BNP, 862 pg/ml. Echocardiogram showed the interventricular septum curved toward the left

Prognostic predictive value of gene mutations in Japanese patients with hypertrophic cardiomyopathy

Although some studies have attempted to find useful prognostic factors in hypertrophic cardiomyopathy (HCM), those results are not fully helpful for use in actual clinical practice. Furthermore, several genetic abnormalities associated with HCM have been identified. However, the genotype–phenotype correlation in HCM remains to be elucidated. Here, we attempted to assess patients with different types of gene mutations causing HCM and investigate the prognosis. A total of 140 patients with HCM underwent a screening test for myofilament gene mutations by direct sequencing of eight sarcomeric genes. Patients with a single mutation in cardiac troponin T, cardiac troponin I, α-tropomyosin, and regulatory and essential light chains were excluded from the study because the number of cases was too small. The clinical presentations and outcomes of the remaining 127 patients with HCM, 31 β-myosin heavy chain (MYH7) mutation carriers, 19 cardiac myosin-binding protein C (MYBPC3) mutation carriers, and 77 mutation non-carriers were analyzed retrospectively. MYBPC3 mutation carriers had a high frequency of ventricular arrhythmia and syncope. Kaplan–Meier curves revealed no significant difference in prognosis among the three groups, but a lack of family history of sudden death (SD) and a past history of syncope were significantly related to poor prognosis. An absence of family history of SD and past history of syncope are useful prognostic factors in patients with HCM. MYH7 and MYBPC3 mutations did not significantly influence prognosis compared to non-carriers. However, patients with the MYBPC3 mutation should be closely followed for the possibility of SD.

dup(8p)/del(8q) recombinant chromosome in a girl with hepatic focal nodular hyperplasia†

A 15‐year‐old girl had exertion dyspnea, focal nodular hyperplasia of the liver, portal vein hypoplasia, portopulmonary hypertension, mental retardation, and minor facial abnormalities. Cytogenetic analysis demonstrated an abnormal chromosome 8 with 8p22‐pter duplication and 8q24.3‐qter deletion, with the duplicated 8p segment attached to band 8q24.3. Her mother had a pericentric inversion of chromosome 8, inv(8)(p22q24.3). Therefore, the girls abnormal chromosome 8 was a recombinant of maternal inversion chromosome: 46,XX,rec(8)dup(8p)inv(8)(p22q24.3)mat. Further characterization of the recombinant chromosome, using array CGH and regional FISH analyses, defined 15 Mb distal 8p duplication and 0.5 Mb 8q deletion. Possible correlation of the recombinant chromosome and hepatic focal nodular hyperplasia in the patient is discussed.

ACVRL1 gene variant in a patient with vein of Galen aneurysmal malformation

Although mutations in the RASA1 gene in vein of Galen aneurysmal malformation (VGAM) and an endoglin gene mutation in a VGAM patient with a family history of hereditary hemorrhagic telangiectasia (HHT) have been identified, most VGAM cases have no mutation in these genes. We sought to detect mutations in other genes related to HHT. We screened for mutations in RASA1 and three genes (endoglin, activin receptor-like kinase 1 (ACVRL1), encoding ALK1, and SMAD4) related to HHT in four VGAM patients. One variant (c.652 C>T p.R218W) in ACVRL1 was identified. Immunoblotting revealed that the ALK1-R218W protein could not promote SMAD1/5/8 phosphorylation by BMP9 stimulation. On the other hand, wild-type ALK1 could enhance the phosphorylation as expected. Furthermore, the transcriptional activation of ALK1-R218W was less efficient than that of wild-type ALK1. We identified 1 variant in ACVRL1 in a VGAM patient. These findings suggest that the ACVRL1 variant-R218W may be associated with the pathogenesis of VGAM.

Maternal mosaicism of an ANKH mutation in a family with craniometaphyseal dysplasia

Craniometaphyseal dysplasia (CMD; OMIM 123000) is a rare genetic disorder characterized by hyperostosis of the craniofacial bones and metaphyseal flaring of the long bones. Patients with CMD often suffer from neurological symptoms due to obstruction of the cranial foramina. CMD is inherited as an autosomal dominant trait, or occurs sporadically with apparent de novo mutations in ANKH, which encodes a putative pyrophosphatetransporting membrane protein. We herein report a family with a daughter affected with CMD who had an ANKH mutation and healthy parents. A parental gene analysis indicated that there was maternal somatic mosaicism for an ANKH mutation. A 7-month-old girl was brought to hospital because of sudden cardiopulmonary arrest. She had right facial nerve palsy from birth, but she had no retardation of her growth and development. She was the first child of healthy Japanese parents with no consanguinity. She was successfully resuscitated in the hospital, but later developed severe hypoxic encephalopathy. The diagnosis of CMD was made based on the clinical expression (hypertelorism, frontonasal bossing, facial nerve palsy and bony choanal stenosis) and radiographs of the cranial and long bones (Fig. 1a–c). To identify the ANKH mutation in this patient, we performed gene analysis using a blood sample. The DNA extracted from white blood cells was polymerase chain reaction (PCR) amplified and sequenced for all 12 exons of the ANKH gene. The primer sets used in this study have been described in an earlier study. This sequence analysis indicated a heterozygous mutation in exon 9 (c.1123_1125delTCC, Ser375del; Fig. 1d), which is a known pathogenic mutation in CMD. The parental DNA from blood cells was subsequently analyzed for ANKH, and electropherograms of exon 9 from the mother showed low peaks of the mutant sequence, suggesting mosaicism of the wild-type and mutant alleles (Fig. 1d). We further evaluated the mosaicism by counting the wild-type alleles and the mutant delTCC alleles after TA cloning of the PCR products. Blood cells, the buccal swab, and nail sample contained 26.9%, 31.8% and 1.7% of the mutant delTCC allele, respectively (Fig. 1e). The effect of somatic mosaicism of the ANKH mutation was not apparent in the mother; hypertelorism or paranasal bossing was not seen, and the chest X-ray examined previously showed no abnormal findings of the clavicles or ribs. Although the ova of the mother were not analyzed in this study, the identical c.1123_1125delTCC mutation in ANKH found in both the mother and the affected daughter indicated that the mother possessed the mutation in her germ cells in a mosaic state, suggesting a recurrence risk for subsequent offspring. All of the previously identified mutations of ANKH were in-frame deletions of amino acids, an insertion of one amino acid due to a splicing defect, and a substitution of an amino acid due to a point mutation (Table 1). Recently, a case of a complex missense mutation and a 12 bp deletion was reported. Of note, two ANKH mutations in exon 9 (Ser375del and Phe377del) are supposed to be hot spot mutations in CMD, because both mutations were found in a total of four families reported so far (Table 1), with Ser375del being found in the present patient. The sequences of ANKH c.1120_1131 consist of short tandem repeats of (TTC) (TCC) (TTC) (TTC), so the slipped strand mispairing during DNA replication may cause the 3 bp deletion of ANKH hot spot mutations. CMD is a rare genetic disorder, and the prevalence and recurrence risk are unknown. To the best of our knowledge, all of the non-familial cases of CMD were sporadic or de novo occurrences, and the possibility of parental mosaicism has not been discussed. Analysis of the present CMD family suggested the presence of a maternal mosaicism in an ANKH mutation, and the mother who was mosaic for the ANKH mutation had no apparent clinical or radiological features of CMD. Parental mosaicism may account for some apparently sporadic or new cases of CMD, and has important implications in the genetic counseling of families affected by CMD. Correspondence: Tamaki Kato, MD, Department of Pediatrics, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Email: drpe059@ndmc.ac.jp Received 12 December 2012; revised 10 January 2013; accepted 5 February 2013. bs_bs_banner

Cardiopulmonary Arrest Caused by Craniometaphyseal Dysplasia

Craniometaphyseal dysplasia is a rare congenital disorder that can cause craniofacial skeleton and tubular bone anomalies. A 7-month-old girl, with congenital facial palsy, mouth breathing, and nasal obstruction, was brought to the hospital because she suffered sudden cardiopulmonary arrest. Computed tomography showed bony narrowing of the choanae. The diagnosis was cardiopulmonary arrest triggered by asphyxia caused by severe choanal stenosis. She showed hypoxic encephalopathy and underwent a tracheotomy and an operation to enlarge choanae. When a neonate or young infant shows signs of nasal obstruction, physicians should aggressively search for and consider the possibility of choanal atresia or severe stenosis.

Rapidly progressive encephalopathy with coagulation abnormality (Case Presentation).

The Discussion and Diagnosis can be found on page 1908. CASE A 19-month-old girl with a history of a cough for several days was referred to the hospital after experiencing status epilepticus. She had been febrile for several hours. She had been healthy before admission and had no history of seizure or exanthema subitum. She was vaccinated with Bacillus Calmette–Guerin vaccine without complications. She was the second child of unrelated healthy parents. Her generalized clonic seizure lasted approximately 1 h and required intravenous diazepam and phenytoin before termination. Endotracheal intubation was performed because she developed a deep coma and severe respiratory distress. Her body temperature was 40 C, and tachycardia, tachypnea and hypertension were seen. There was no nuchal rigidity, and the bilateral papillary light reflex was intact. She showed laboured respiration, but all other findings of the physical examination were unremarkable. A complete blood cell count revealed an elevated leucocyte count (13.3 · 10 ⁄ L with 46.5% neutrophils) and normal levels of haemoglobin (11.6 g ⁄ dL) and platelet (299 · 10 ⁄ L). Hyperglycaemia (205 mg ⁄ dL) was seen. Serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, sodium, potassium, chloride, blood urea nitrogen, creatinine and creatine kinase were all within normal limits. A venous blood gas analysis showed mild metabolic acidosis