Hirohito Shima

Systematic molecular analyses of SHOX in Japanese patients with idiopathic short stature and Leri-Weill dyschondrosteosis

The etiology of idiopathic short stature (ISS) and Leri–Weill dyschondrosteosis (LWD) in European patients is known to include SHOX mutations and copy-number variations (CNVs) involving SHOX and/or the highly evolutionarily conserved non-coding DNA elements (CNEs) flanking the gene. However, the frequency and types of SHOX abnormalities in non-European patients and the clinical importance of mutations in the CNEs remains to be clarified. Here, we performed systematic molecular analyses of SHOX for 328 Japanese patients with ISS or LWD. SHOX abnormalities accounted for 3.8% of ISS and 50% of LWD cases. CNVs around SHOX were identified in 16 cases, although the ~47u2009kb deletion frequently reported in European patients was absent in our cases. Probably damaging mutations and benign/silent substitutions were detected in four cases, respectively. Although CNE-linked substitutions were detected in 15 cases, most of them affected poorly conserved nucleotides and were shared by unaffected individuals. These results suggest that the frequency and mutation spectrum of SHOX abnormalities are comparable between Asian and European patients, with the exception of a European-specific downstream deletion. Furthermore, this study highlights the clinical importance and genetic heterogeneity of the SHOX-flanking CNVs, and indicates a limited clinical significance of point mutations in the CNEs.

Poor outcome with hematopoietic stem cell transplantation for bone marrow failure and MDS with severe MIRAGE syndrome phenotype

Key Points Success of hematopoietic stem cell transplantation for MIRAGE syndrome may be limited by syndrome-specific comorbidities. SAMD9 mutations associated with MIRAGE syndrome are a newly described cause of congenital amegakaryocytic thrombocytopenia.

Xp22.31 Microdeletion due to Microhomology-Mediated Break-Induced Replication in a Boy with Contiguous Gene Deletion Syndrome

The Xp22.31 region is characterized by a low frequency of interspersed repeats and a low GC content. Submicroscopic deletions at Xp22.31 involving STS and ANOS1 (alias KAL1) underlie X-linked ichthyosis and Kallmann syndrome, respectively. Of the known microdeletions at Xp22.31, a common approximately 1.5-Mb deletion encompassing STS was ascribed to nonallelic homologous recombination, while 2 ANOS1-containing deletions were attributed to nonhomologous end-joining. However, the genomic bases of other microdeletions within the Xp22.31 region remain to be elucidated. Here, we identified a 2,735,696-bp deletion encompassing STS and ANOS1 in a boy with X-linked ichthyosis and Kallmann syndrome. The breakpoints of the deletion were located within Alu repeats and shared 2-bp microhomology. The fusion junction was not associated with nucleotide stretches, and the breakpoint-flanking regions harbored no palindromes or noncanonical DNA motifs. These results indicate that microhomology-mediated break-induced replication (MMBIR) can cause deletions at Xp22.31, resulting in contiguous gene deletion syndrome. It appears that interspersed repeats without other known rearrangement-inducing DNA features or high GC contents are sufficient to stimulate MMBIR at Xp22.31.

Complex X-Chromosomal Rearrangements in Two Women with Ovarian Dysfunction: Implications of Chromothripsis/Chromoanasynthesis-Dependent and -Independent Origins of Complex Genomic Alterations.

Our current understanding of the phenotypic consequences and the molecular basis of germline complex chromosomal rearrangements remains fragmentary. Here, we report the clinical and molecular characteristics of 2 women with germline complex X-chromosomal rearrangements. Patient 1 presented with nonsyndromic ovarian dysfunction and hyperthyroidism; patient 2 exhibited various Turner syndrome- associated symptoms including ovarian dysfunction, short stature, and autoimmune hypothyroidism. The genomic abnormalities of the patients were characterized by array-based comparative genomic hybridization, high-resolution karyotyping, microsatellite genotyping, X-inactivation analysis, and bisulfite sequencing. Patient 1 carried a rearrangement of unknown parental origin with a 46,X,der(X)(pter→ p22.1::p11.23→q24::q21.3→q24::p11.4→pter) karyotype, indicative of a catastrophic chromosomal reconstruction due to chromothripsis/chromoanasynthesis. Patient 2 had a paternally derived isochromosome with a 46,X,der(X)(pter→ p22.31::q22.1→q10::q10→q22.1::p22.31→pter) karyotype, which likely resulted from 2 independent, sequential events. Both patients showed completely skewed X inactivation. CpG sites at Xp22.3 were hypermethylated in patient 2. The results indicate that germline complex X-chromosomal rearrangements underlie nonsyndromic ovarian dysfunction and Turner syndrome. Disease-causative mechanisms of these rearrangements likely include aberrant DNA methylation, in addition to X-chromosomal mispairing and haploinsufficiency of genes escaping X inactivation. Notably, our data imply that germline complex X-chromosomal rearrangements are created through both chromothripsis/chromoanasynthesis-dependent and -independent processes.

Two patients with MIRAGE syndrome lacking haematological features: role of somatic second-site reversion SAMD9 mutations

Background Myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (MIRAGE) syndrome is a recently described congenital disorder caused by heterozygous SAMD9 mutations. The phenotypic spectrum of the syndrome remains to be elucidated. Methods and results We describe two unrelated patients who showed manifestations compatible with MIRAGE syndrome, with the exception of haematological features. Leucocyte genomic DNA samples were analysed with next-generation sequencing and Sanger sequencing, revealing the patients to have two de novoSAMD9 mutations on the same allele (patient 1u2009p.[Gln695*; Ala722Glu] and patient 2u2009p.[Gln39*; Asp769Gly]). In patient 1, p.Gln695* was absent in genomic DNA extracted from hair follicles, implying that the non-sense mutation was acquired somatically. In patient 2, with the 46,XX karyotype, skewed X chromosome inactivation pattern was found in leucocyte DNA, suggesting monoclonality of cells in the haematopoietic system. In vitro expression experiments confirmed the growth-restricting capacity of the two missense mutant SAMD9 proteins that is a characteristic of MIRAGE-associated SAMD9 mutations. Conclusions Acquisition of a somatic nonsense SAMD9 mutation in the cells of the haematopoietic system might revert the cellular growth repression caused by the germline SAMD9 mutations (ie, second-site reversion mutations). Unexpected lack of haematological features in the two patients would be explained by the reversion mutations.

SOX2 nonsense mutation in a patient clinically diagnosed with non-syndromic hypogonadotropic hypogonadism

Hypogonadotropic hypogonadism (HH) is a genetically heterogeneous condition that occurs either as an isolated disorder or as a component of congenital malformation syndromes. SOX2 is a causative gene of syndromic HH characterized by anophthalmia, microphthalmia, or coloboma and other neurological defects such as epilepsy. To date, the causal relationship between SOX2 abnormalities and non-syndromic HH remains speculative. Here, we identified a nonsense mutation of SOX2 in a male patient clinically diagnosed with non-syndromic HH. The patient had epilepsy but no additional clinical features. Ophthalmological examination revealed no abnormalities except for decreased thickness of the retinal nerve fiber layer. Audiometry showed mild sensorineural hearing impairment of both ears. Hormonal evaluation suggested isolated gonadotropin deficiency. Next-generation sequencing-based mutation screening of 13 major causative genes for HH identified a p.Lys35∗ mutation in SOX2 and excluded pathogenic mutations in other tested genes. The p.Lys35∗ mutation appeared to encode a non-functioning SOX2 protein that lacks 283 of 317 amino acids. The SOX2 mutation was absent in the maternal DNA sample, while a paternal sample was unavailable for sequence analysis. These results expand the clinical consequences of SOX2 haploinsufficiency to include non-syndromic HH. Systematic mutation screening using a next-generation sequencer and detailed evaluation of nonspecific ocular/neurological features may help identify SOX2 mutation-positive individuals among HH patients.

NR0B1 Frameshift Mutation in a Boy with Idiopathic Central Precocious Puberty.

NR0B1 is the causative gene for X-linked adrenal hypoplasia congenita, characterized by adrenal insufficiency, hypogonadotropic hypogonadism, and infertility. We identified an NR0B1 frameshift mutation in a boy with precocious puberty who had no signs of adrenal insufficiency. Blood examination revealed elevated testosterone levels and gonadotropin hyperresponses to gonadotropin releasing hormone (GnRH) stimulation, together with normal adrenal hormone levels. GnRH analog treatment partially ameliorated his clinical features. Molecular analysis identified a p.Glu3fsAla*16 in NR0B1. These results expand the clinical manifestations of NR0B1 mutations to include central precocious puberty without adrenal insufficiency. NR0B1 mutations likely underlie androgen overproduction via GnRH-dependent and -independent mechanisms.

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)

An unclassified variant of CHD7 activates a cryptic splice site in a patient with CHARGE syndrome

CHARGE syndrome is a rare autosomal dominant disease that is typically caused by heterozygous CHD7 mutations. A de novo variant in a CHD7 splicing acceptor site (NM_017780.3:c.7165–4A>G) was identified in a Japanese boy with CHARGE syndrome. This variant has been considered to be an “unclassified variant” due to its position outside the consensus splicing sites. In this study, abnormal splicing derived from this known variant was confirmed by cDNA sequencing.

Germline loss of function SAMD9 and SAMD9L alterations in adult myelodysplastic syndromes

TO THE EDITOR:nnFamilial bone marrow failure (BMF) syndromes present typically in children and younger adults.[1][1][⇓][2]-[3][3] A number of germline (GL) mutations in genes such as DDX41 ,[4][4] RUNX1 ,[5][5] ETV6 ,[6][6] GATA2 ,[7][7] and ANKRD26 [8][8] have been implicated in the pathogenesis