Naoko Amano

SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7

Adrenal hypoplasia is a rare, life-threatening congenital disorder. Here we define a new form of syndromic adrenal hypoplasia, which we propose to term MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) syndrome. By exome sequencing and follow-up studies, we identified 11 patients with adrenal hypoplasia and common extra-adrenal features harboring mutations in SAMD9. Expression of the wild-type SAMD9 protein, a facilitator of endosome fusion, caused mild growth restriction in cultured cells, whereas expression of mutants caused profound growth inhibition. Patient-derived fibroblasts had restricted growth, decreased plasma membrane EGFR expression, increased size of early endosomes, and intracellular accumulation of giant vesicles carrying a late endosome marker. Of interest, two patients developed myelodysplasitc syndrome (MDS) that was accompanied by loss of the chromosome 7 carrying the SAMD9 mutation. Considering the potent growth-restricting activity of the SAMD9 mutants, the loss of chromosome 7 presumably occurred as an adaptation to the growth-restricting condition.

Heterozygous Mutations in Natriuretic Peptide Receptor-B (NPR2) Gene as a Cause of Short Stature in Patients Initially Classified as Idiopathic Short Stature

CONTEXT Based on the stature observed in relatives of patients with acromesomelic dysplasia, type Maroteaux, homozygous for mutations in natriuretic peptide receptor B gene (NPR2), it has been suggested that heterozygous mutations in this gene could be responsible for the growth impairment observed in some children with idiopathic short stature (ISS). OBJECTIVE The objective of the study was to investigate the presence of NPR2 mutations in a group of patients with ISS. PATIENTS AND METHODS The NPR2 coding region was directly sequenced in 47 independent patients with ISS. The functional consequences of NPR2 nonsynonymous variations were established using in vitro cell-based assays. RESULTS Three novel heterozygous NPR2 mutations were identified: c.226T>C (p.Ser76Pro), c.788G>C (p.Arg263Pro), and c.2455C>T (p.Arg819Cys). These allelic variants were not found in our controls or in the 1000 Genomes database. In silico analysis suggested that the three missense mutations are probably damaging. All of them were selected for in vitro functional evaluation. Cells transfected with the three mutants failed to produce cyclic GMP after treatment with C-type natriuretic peptide. Cells cotransfected with mutant and wild-type-NPR-B (1:1) showed a significant decrease in cGMP levels after C-type natriuretic peptide stimulation in comparison with cells cotrasnfected with empty vector and wild type, suggesting a dominant-negative effect. These three mutations segregated with short stature phenotype in an autosomal dominant pattern (height SD score ranged from -4.5 to -1.7). One of these patients and two relatives have disproportionate short stature, whereas in another patient a nonspecific skeletal abnormality was observed. All three of these patients were treated with recombinant human GH (33-50 μg/kg · d) without significant height SD score change during therapy. CONCLUSIONS We identified heterozygous NPR2 mutations in 6% of patients initially classified as ISS. Affected patients have mild and variable degrees of short stature without a distinct phenotype. Heterozygous mutations in NPR2 could be an important cause of nonsyndromic familial short stature.

Identification and Functional Characterization of Two Novel NPR2 Mutations in Japanese Patients With Short Stature

CONTEXT C-type natriuretic peptide-natriuretic peptide receptor B (NPR-B) signaling is critical for endochondral ossification, which is responsible for longitudinal growth in limbs and vertebrae. Biallelic NPR2 mutations cause acromesomelic dysplasia, type Maroteaux, which is bone dysplasia characterized by severe short stature and short limbs. A monoallelic NPR2 mutation has been suggested to mildly impair long bone growth. OBJECTIVE The goal of this study was to identify and characterize NPR2 mutations among Japanese patients with short stature. SUBJECTS AND METHODS We enrolled 101 unrelated Japanese patients with short stature. NPR2 and NPPC were sequenced, and the identified variants were characterized in vitro. RESULTS In two subjects, we identified two novel heterozygous NPR2 mutations (R110C and Q417E) causing a loss of C-type natriuretic peptide-dependent cGMP generation capacities and having dominant-negative effects. R110C was defective in trafficking from the endoplasmic reticulum to the Golgi apparatus. In contrast, Q417E showed clear cell surface expression. CONCLUSIONS We identified heterozygous NPR2 mutations in 2% of Japanese patients with short stature. Our in vitro findings indicate that NPR2 mutations have a dominant negative effect, and their dominant-negative mechanisms vary corresponding to the molecular pathogenesis of the mutations.

A Novel Mutation in LEPRE1 That Eliminates Only the KDEL ER- Retrieval Sequence Causes Non-Lethal Osteogenesis Imperfecta

Prolyl 3-hydroxylase 1 (P3H1), encoded by the LEPRE1 gene, forms a molecular complex with cartilage-associated protein (CRTAP) and cyclophilin B (encoded by PPIB) in the endoplasmic reticulum (ER). This complex is responsible for one step in collagen post-translational modification, the prolyl 3-hydroxylation of specific proline residues, specifically α1(I) Pro986. P3H1 provides the enzymatic activity of the complex and has a Lys-Asp-Glu-Leu (KDEL) ER-retrieval sequence at the carboxyl terminus. Loss of function mutations in LEPRE1 lead to the Pro986 residue remaining unmodified and lead to slow folding and excessive helical post-translational modification of type I collagen, which is seen in both dominant and recessive osteogenesis imperfecta (OI). Here, we present the case of siblings with non-lethal OI due to novel compound heterozygous mutations in LEPRE1 (c.484delG and c.2155dupC). The results of RNA analysis and real-time PCR suggest that mRNA with c.2155dupC escapes from nonsense-mediated RNA decay. Without the KDEL ER- retrieval sequence, the product of the c.2155dupC variant cannot be retained in the ER. This is the first report of a mutation in LEPRE1 that eliminates only the KDEL ER-retrieval sequence, whereas other functional domains remain intact. Our study shows, for the first time, that the KDEL ER- retrieval sequence is essential for P3H1 functionality and that a defect in KDEL is sufficient for disease onset.

The Effect of Intramuscular Testosterone Enanthate Treatment on Stretched Penile Length in Prepubertal Boys With Hypospadias

OBJECTIVES To define the responses of stretched penile length (PL) to intramuscular testosterone enanthate (TE) treatment in prepubertal boys with hypospadias. METHODS We examined 17 Japanese boys with hypospadias at 1.4 +/- 1.3 (mean +/- SD) years of age. Their PLs were 2.79 +/- 0.37 cm and -1.16 +/- 0.88 SD of the mean. The etiology of hypospadias included sex chromosome disorders of sex development in 5, mastermind-like domain-containing 1 deficiency in 1, and unknown cause in the others. No mutation was identified in the SRD5A2 or AR gene. All the boys received as many as three intramuscular injections of TE 25 mg every 4 weeks (one injection in 3 boys, two in 5, and three in 9). RESULTS The TE treatment significantly increased PL by 1.01 +/- 0.50 cm and 2.27 +/- 0.99 SD (cm, P = .0002; SD, P = .0002). Age, body surface area (BSA), and PL before the treatment did not significantly correlate with the effect of TE treatment on PL. The effect of TE treatment on PL at the first injection in Japanese boys with hypospadias (0.35 +/- 0.20 cm and 0.91 +/- 0.62 SD) was significantly less than that in micropenis at 2.6 +/- 3.1 years of age (0.64 +/- 0.26 cm and 1.37 +/- 0.68 SD) (cm, P = .0008; SD, P = .02). CONCLUSIONS These data indicate that (1) the intramuscular TE treatment significantly increases PL for hypospadias in prepubertal boys, with no demonstrable SRD5A2 or AR mutation; (2) age, BSA, and PL before the treatment are not significantly contributing factors to the effect of TE treatment; and (3) the effect of TE treatment for hypospadias is significantly less than that for micropenis.

Gradual Loss of ACTH Due to a Novel Mutation in LHX4: Comprehensive Mutation Screening in Japanese Patients with Congenital Hypopituitarism

Mutations in transcription factors genes, which are well regulated spatially and temporally in the pituitary gland, result in congenital hypopituitarism (CH) in humans. The prevalence of CH attributable to transcription factor mutations appears to be rare and varies among populations. This study aimed to define the prevalence of CH in terms of nine CH-associated genes among Japanese patients. We enrolled 91 Japanese CH patients for DNA sequencing of POU1F1, PROP1, HESX1, LHX3, LHX4, SOX2, SOX3, OTX2, and GLI2. Additionally, gene copy numbers for POU1F1, PROP1, HESX1, LHX3, and LHX4 were examined by multiplex ligation-dependent probe amplification. The gene regulatory properties of mutant LHX4 proteins were characterized in vitro. We identified two novel heterozygous LHX4 mutations, namely c.249-1G>A, p.V75I, and one common POU1F1 mutation, p.R271W. The patient harboring the c.249-1G>A mutation exhibited isolated growth hormone deficiency at diagnosis and a gradual loss of ACTH, whereas the patient with the p.V75I mutation exhibited multiple pituitary hormone deficiency. In vitro experiments showed that both LHX4 mutations were associated with an impairment of the transactivation capacities of POU1F1 andαGSU, without any dominant-negative effects. The total mutation prevalence in Japanese CH patients was 3.3%. This study is the first to describe, a gradual loss of ACTH in a patient carrying an LHX4 mutation. Careful monitoring of hypothalamic–pituitary -adrenal function is recommended for CH patients with LHX4 mutations.

IMAGe syndrome: clinical and genetic implications based on investigations in three Japanese patients

Arboleda et al. have recently shown that IMAGe (intra‐uterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital abnormalities) syndrome is caused by gain‐of‐function mutations of maternally expressed gene CDKN1C on chromosome 11p15.5. However, there is no other report describing clinical findings in patients with molecularly studied IMAGe syndrome. Here, we report clinical and molecular findings in Japanese patients.

Radiological evolution in IMAGe association: A case report

IMAGe association is a recently recognized multi‐system disorder of unknown etiology. IMAGe is a mnemonic acronym that stands for Intrauterine growth retardation, Metaphyseal dysplasia, Adrenal hypoplasia congenita, and Genital anomalies (OMIM 300290). Suspicion for the disorder is readily raised by the distinctive clinical and endocrinological constellation, and radiological identification of metaphyseal dysplasia is crucial for the diagnosis. However, knowledge of the onset, evolution, severity, and variation of the metaphyseal dysplasia is currently limited. We illustrate the radiological evolution of an affected girl from her premature birth to early childhood. Her initial skeletal changes included thin ribs, delayed ossification of the juxtatruncal bones, and delayed epiphyseal ossification. The former two became less conspicuous during infancy. Metaphyseal dysplasia was not discerned at birth. However, mild metaphyseal cupping, sclerosis and longitudinal striations became manifest in late infancy, and then progressed with age. It is thought that the skeletal alterations in IMAGe association encompass retarded endochondral ossification normalized later on and mild metaphyseal dysplasia of postnatal onset.

Genetic defects in pediatric-onset adrenal insufficiency in Japan

CONTEXT Most patients with pediatric-onset primary adrenal insufficiency (PAI), such as 21-hydroxylase deficiency, can be diagnosed by measuring the urine or serum levels of steroid metabolites. However, the etiology is often difficult to determine in a subset of patients lacking characteristic biochemical findings. OBJECTIVE To assess the frequency of genetic defects in Japanese children with biochemically uncharacterized PAI and characterize the phenotypes of mutation-carrying patients. METHODS We enrolled 63 Japanese children (59 families) with biochemically uncharacterized PAI, and sequenced 12 PAI-associated genes. The pathogenicities of rare variants were assessed based on in silico analyses and structural modeling. We calculated the proportion of mutation-carrying patients according to demographic characteristics. RESULTS We identified genetic defects in 50 (85%) families: STAR in 19, NR0B1 in 18, SAMD9 in seven, AAAS in two, NNT in two, MC2R in one and CDKN1C in one. NR0B1 defects were identified in 78% of the male patients that received both glucocorticoid and mineralocorticoid replacement therapy and had normal male external genitalia. STAR defects were identified in 67% of female and 9% of male patients. Seven of the 19 patients with STAR defects developed PAI at age two or older, out of whom, five did not have mineralocorticoid deficiency. CONCLUSIONS Molecular testing elucidated the etiologies of most biochemically uncharacterized PAI patients. Genetic defects such as NR0B1 defects are presumed based on phenotypes, while others with broad phenotypic variability, such as STAR defects, are difficult to diagnose. Molecular testing is a rational approach to diagnosis in biochemically uncharacterized PAI patients.

Comment on: Acquired monosomy 7 myelodysplastic syndrome in a child with clinical features of dyskeratosis congenita and IMAGe association

To the Editor In a 2010 article in this journal, we reported a case of acquired monosomy 7 myelodysplastic syndrome (MDS) in a male with congenital adrenocortical insufficiency, genital anomalies, growth delay, and chronic lungdisease.1 Thegenetic basis for his conditionwasunclear at the time. In follow-up studies,wehavedetermined that hehasMIRAGE syndrome (OMIM 617053),2 a newly recognized multisystem disorder caused by heterozygous gain-of-function mutations in SAMD9, a ubiquitously expressed gene located on the long arm of chromosome 7. SAMD9 encodes a facilitator of endosome fusion that functions as a growth suppressor.2,3 Clinical hallmarks ofMIRAGE syndrome include myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy.2,4 Following institutional ethics committee approval and written informed consent from the family, buccal cell DNA was collected from the patient and sequenced. A novel heterozygous SAMD9 sequence variant (c.2691A>G, p.Ile897Met) was identified. To study the impact of the Ile897Met variant on cell proliferation, HEK293 cells were stably transfectedwith a doxycycline-inducible construct encoding either wild-type or mutant SAMD9, using an established method.2 Enforced expression of wild-type SAMD9 resulted in a modest decrease in cell proliferation, consistent with its known role as a growth suppressor (Fig. 1A). Expression of the Ile897Met mutant caused a marked reduction in cell growth, confirming that this is a pathogenic variant (Fig. 1B). This expands the list of germline SAMD9mutations linked to MIRAGE syndrome or isolatedMDS.2,4,5 Studies of other patients have shown that somatic mutations or chromosomal loss can modify SAMD9 phenotypes in humans.2,4,5 The development of monosomy 7 MDS in the setting of MIRAGE syndrome is a form of adaptation by aneuploidy; the copy of chromosome 7 harboring the mutant SAMD9 allele is preferentially lost from hematopoietic progenitor cells, thereby conferring a clonal growth advantage. MIRAGE syndrome is associatedwith a highmortality in early childhood, due mainly to an increased susceptibility to severe infections.2,4 Our patient, who underwent a reduced intensity allogeneic peripheral blood stem cell transplant (PBSCT) at age 6 (∼10 years ago), is a long-term survivor. He has had recurrent respiratory infections that progressed to bronchiectasis, and he has required treatment with systemic and inhaled antibiotics for chronic endobronchitis with pulmonary exacerbations. His other long-term medical issues include (i) growth delay (height and weight in the first centile), (ii)