Maki Fukami

Pseudoautosomal deletions encompassing a novel homeobox gene cause growth failure in idiopathic short stature and Turner syndrome

Growth retardation resulting in short stature is a major concern for parents and due to its great variety of causes, a complex diagnostic challenge for clinicians. A major locus involved in linear growth has been implicated within the pseudoautosomal region (PAR1) of the human sex chromosomes. We have determined an interval of 170 kb of DNA within PAR1 which was deleted in 36 individuals with short stature and different rearrangements on Xp22 or Yp11.3. This deletion was not detected in any of the relatives with normal stature or in a further 30 individuals with rearrangements on Xp22 or Yp11.3 with normal height. We have isolated a homeobox-containing gene (SHOX} from this region, which has at least two alternatively spliced forms, encoding proteins with different patterns of expression. We also identified one functionally significant SHOX mutation by screening 91 individuals with idiopathic short stature. Our data suggest an involvement of SHOX in idiopathic growth retardation and in the short stature phenotype of Turner syndrome patients.

The IG-DMR and the MEG3-DMR at human chromosome 14q32.2: hierarchical interaction and distinct functional properties as imprinting control centers.

Human chromosome 14q32.2 harbors the germline-derived primary DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived secondary MEG3-DMR, together with multiple imprinted genes. Although previous studies in cases with microdeletions and epimutations affecting both DMRs and paternal/maternal uniparental disomy 14-like phenotypes argue for a critical regulatory function of the two DMRs for the 14q32.2 imprinted region, the precise role of the individual DMR remains to be clarified. We studied an infant with upd(14)pat body and placental phenotypes and a heterozygous microdeletion involving the IG-DMR alone (patient 1) and a neonate with upd(14)pat body, but no placental phenotype and a heterozygous microdeletion involving the MEG3-DMR alone (patient 2). The results generated from the analysis of these two patients imply that the IG-DMR and the MEG3-DMR function as imprinting control centers in the placenta and the body, respectively, with a hierarchical interaction for the methylation pattern in the body governed by the IG-DMR. To our knowledge, this is the first study demonstrating an essential long-range imprinting regulatory function for the secondary DMR.

CXorf6 is a causative gene for hypospadias

46,XY disorders of sex development (DSD) refer to a wide range of abnormal genitalia, including hypospadias, which affects ∼0.5% of male newborns. We identified three different nonsense mutations of CXorf6 in individuals with hypospadias and found that its mouse homolog was specifically expressed in fetal Sertoli and Leydig cells around the critical period for sex development. These data imply that CXorf6 is a causative gene for hypospadias.

Silver-Russell syndrome in a girl born after in vitro fertilization: partial hypermethylation at the differentially methylated region of PEG1/MEST

AbstractPurpose: The prevalence of low birth weight (LBW) is increased in subjects born after assisted reproduction technology (ART), and defective imprinting has frequently been identified in patients with Beckwith-Wiedermann and Angelman syndromes conceived by ART. Thus, we examined methylation pattern in a girl born after ART who had Silver-Russell syndrome (SRS) which can be caused by maternal uniparental disomy for chromosome 7 and by hypomethylation of the differentially methylated region (DMR) of H19. Methods: We examined methylation status of 31 cytosines at the CpG dinucleotides in the DMR of PEG1/MEST on 7q32.2 and 23 cytosines at the CpG dinucleotides in the DMR of H19 on 11p15, using leukocyte genomic DNA. Results: Eight of the 31 cytosines in the patient and four of the 31 cytosines in the father were hypermethylated in the PEG1/MEST-DMR. In the H19-DMR, no abnormal methylation pattern was identified in the patient. Conclusion: The results suggest that hypermethylation of paternally expressed genes including PEG1/MEST, which usually have growth-promoting effects, may be relevant to LBW in subjects conceived by ART.

Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system

Introducing a point mutation is a fundamental method used to demonstrate the roles of particular nucleotides or amino acids in the genetic elements or proteins, and is widely used in in vitro experiments based on cultured cells and exogenously provided DNA. However, the in vivo application of this approach by modifying genomic loci is uncommon, partly due to its technical and temporal demands. This leaves many in vitro findings un-validated under in vivo conditions. We herein applied the CRISPR/Cas9 system to generate mice with point mutations in their genomes, which led to single amino acid substitutions in proteins of interest. By microinjecting gRNA, hCas9 mRNA and single-stranded donor oligonucleotides (ssODN) into mouse zygotes, we introduced defined genomic modifications in their genome with a low cost and in a short time. Both single gRNA/WT hCas9 and double nicking set-ups were effective. We also found that the distance between the modification site and gRNA target site was a significant parameter affecting the efficiency of the substitution. We believe that this is a powerful technique that can be used to examine the relevance of in vitro findings, as well as the mutations found in patients with genetic disorders, in an in vivo system.

OTX2 Mutation in a Patient with Anophthalmia, Short Stature, and Partial Growth Hormone Deficiency: Functional Studies Using the IRBP, HESX1, and POU1F1 Promoters

CONTEXT OTX2 is a transcription factor gene essential for eye development. Although recent studies suggest the involvement of OTX2 in pituitary function, there is no report demonstrating a positive role of OTX2 in the pituitary function. OBJECTIVE The objective of the study was to report the results of functional studies indicating the relevance of OTX2 to pituitary function. PATIENT A Japanese female patient with bilateral anophthalmia was found to have short stature (height, -3.3 sd) and isolated partial GH deficiency (peak serum GH 3.1 and 9.7 mug/liter after insulin and arginine stimulations, respectively; serum IGF-I 37 ng/ml) at 3 yr 9 months of age. Magnetic resonance imaging delineated apparently normal pituitary gland. RESULTS Mutation analysis showed a de novo heterozygous frameshift mutation (c.402insC) that is predicted to retain the homeodomain but lose the transactivation domain. Functional studies revealed that the wild-type and mutant OTX2 proteins localized to the nucleus and bound to the target sequences within the IRBP (interstitial retinoid-binding protein), HESX1 (HESX homeobox 1), and POU1F1 promoters. Furthermore, the wild-type OTX2 protein markedly transactivated the promoters of IRBP ( approximately 27-fold), HESX1 ( approximately 4.5-fold), and POU1F1 ( approximately 19-fold), whereas the mutant OTX2 protein barely retained the transactivation activities and had no dominant-negative effects. CONCLUSIONS The results provide direct evidence for OTX2 being involved in the pituitary function. It is likely that the heterozygous severe OTX2 loss-of-function mutation caused GH deficiency and short stature, primarily because of decreased transactivation function for HESX1 and POU1F1.

Cytochrome P450 Oxidoreductase Deficiency in Three Patients Initially Regarded as Having 21-Hydroxylase Deficiency and/or Aromatase Deficiency: Diagnostic Value of Urine Steroid Hormone Analysis

In this study, we report on three Japanese patients with cytochrome P450 oxidoreductase (POR) deficiency (PORD). Case one was a 46,XY patient who was found to have mildly increased 17α-hydroxyprogesterone (17-OHP) by the neonatal mass screening. There was no maternal virilization during pregnancy, and he had no skeletal or genital abnormality. Thus, he was initially diagnosed as having nonclassical 21-hydroxylase deficiency (21-OHD). Cases two and three were 46,XX patients who were identified because of severely virilized external genitalia and maternal virilization during pregnancy. In case two, the neonatal mass screening was normal, and she had no skeletal abnormality except for mild adduction of bilateral third toes. Thus, she was initially diagnosed as having aromatase deficiency. In case three, the neonatal mass screening showed moderately increased 17-OHP, and no skeletal lesion other than rigid second metacarpophalangeal joints was identified in early infancy. Thus, she was initially suspected as having 21-OHD and/or aromatase deficiency. Subsequently, endocrine studies including urine steroid hormone analysis were performed for the assessment of glucocorticoid treatment in case one and for the virilized genitalia in cases two and three, showing adrenal and/or gonadal dysfunction characteristic of PORD. Thus, molecular analysis of POR was carried out, demonstrating homozygosity for R457H in cases one through three. The results imply that clinical features in PORD can be similar to those in 21-OHD or aromatase deficiency, and that comprehensive assessment of the pregnant course, physical examination, and adrenal and gonadal function studies is essential for the precise diagnosis of PORD.

Transactivation Function of an ∼800-bp Evolutionarily Conserved Sequence at the SHOX 3′ Region: Implication for the Downstream Enhancer

To the Editor: In the October 2005 issue of the Journal, Benito-Sanz et al. (2005) reported an association of Leri-Weill dyschondrosteosis (LWD [MIM 127300]) with a novel class of heterozygous pseudoautosomal region 1 (PAR1) deletions downstream of SHOX (short-stature homeobox-containing gene [MIM 312865]) in 12 patients with two copies of intact SHOX coding sequences. The deletions were variable in size, with the smallest region of overlapping deletion (SRO) of ∼30 kb between DXYS10086 and rs7067102. The results—in conjunction with the report of Flanagan et al. (2002) describing a monoallelic SHOX expression in the bone marrow fibroblasts taken from the distal radius of a patient with LWD with two copies of normal SHOX coding exons and hemizygosity for a region around DXYS233 downstream of SHOX—suggest the presence of a downstream enhancer for SHOX transcription around the ∼30-kb SRO. Consistent with this, Fukami et al. (2005) found that (1) a 240–350-kb deletion including DXYS233 is present in a heterozygous status in a mother with LWD and two copies of intact SHOX coding exons and (2) the same deletion is present in a hemizygous status in her daughter with Langer mesomelic dysplasia and a mosaic-ring X chromosome missing the PAR1. Here, we report that the putative SHOX enhancer may reside on an ∼800-bp evolutionarily conserved sequence (ECS). First, we analyzed the SHOX 3′ region in five Japanese families in which the proband and one of the parents had variable degrees of LWD and stature from short to low normal in the presence of two copies of intact SHOX-coding exons. The study was approved by the Institutional Review Board Committee at the National Center for Child Health and Development. Genotyping analysis was performed with primers and methods shown in table 1; results indicate that a deletion between SHOX-SNP792 on the 3′ UTR and DXYS85 was shared by the LWD-affected mother and daughter in family A and that a deletion between rs5946324 and rs4504827 was common to the LWD-affected father and daughter in family B (table 2 and fig. 1A). Furthermore, FISH analysis was performed with an RP13-167H21 BAC probe defining a region from rs5946324 to DXYS233 (Ensembl Genome Browser); results show only a single signal in the mother and the daughter of family A and an obviously different signal intensity in the father and the daughter of family B (fig. 2). The results, together with our previous data (Fukami et al. 2005), imply that an ∼40-kb region between rs5946326 and rs4504827 is the SRO in the Japanese patients (SRO-J) (fig. 1A). The SRO-J is in a close agreement with the ∼30-kb SRO in the white patients (SRO-W) (Benito-Sanz et al. 2005), and a region between rs5946326 and rs7067102 is shared by all the patients with SHOX 3′ deletions (fig. 1A). Figure 1 A, PAR1 deletions in the SHOX downstream region. Top, Pedigrees of families A and B. LWD is exhibited by the mother and the daughter in family A and by the father and the daughter in family B. The height of each subject is expressed as an SD score. Bottom, ... Figure 2 Results of the FISH analysis. Red signals indicate the RP13-167H21 region (thick arrows), and green signals represent DXZ1 (arrowheads) or DYZ3 (thin arrow). The RP13-167H21 region is deleted from one of the two X chromosomes of the mother and the daughter ... Table 1 Primer Sequences and PCR Conditions Used for Genotyping[Note] Table 2 Summary of Polymorphism Analyses[Note] Next, we searched the UCSC Genome Browser for the ECSs within the SROs. Seven ECSs (ECS1–ECS7) were present within the SRO-J, whereas ECS6 and ECS7 were found to reside outside the SRO-W (fig. 1B). ECS3–ECS7 were well conserved in chicken and dog, which preserve Shox, and were absent in mouse and rat, which lack Shox (Clement-Jones et al. 2000; Ensembl Genome Browser). ECS4 was also conserved in Fugu and zebrafish, which preserve Shox. By contrast, ECS1 was absent in chicken, and ECS2 was conserved in mouse and rat. Whereas ECS3 and ECS4 were not described in chimpanzee, the sequence analysis remains poor for the Shox 3′ region in chimpanzee, in contrast to the detailed analysis of that region in chicken and dog. These findings suggest that ECS3–ECS5 can be regarded as candidate regions harboring the putative downstream enhancer. In this regard, since ECS3–ECS5 reside between rs5988437 and rs5946533 (UCSC Genome Browser), they should be deleted from the patients in the three families defining the SRO-J (fig. 1A). Thus, we examined the transcription activity of ECS3–ECS5 as well as ECS6 with the dual-luciferase reporter assay system (Promega). Luciferase reporter constructs containing each ECS (ECS3, 861 bp; ECS4, 824 bp; ECS5, 441 bp; ECS6, 634 bp) inserted into the 3′ region of the luciferase gene were created using the pGL3 vector with the SV40 promoter or the human SHOX promoter on exon 2 (−432 to +5 bp) (Blaschke et al. 2003) (fig. 1B). The U2OS osteosarcoma cell line expressing SHOX (Rao et al. 2001) was transfected using lipofectamine (Invitrogen) with each reporter vector together with the pRL-CMV vector used as an internal control for the transfection, and luciferase assays were performed 36 h later. After the experiments were performed five times, the normalized luciferase activity was found to be significantly increased only when the reporter vector with ECS4 and the SHOX promoter was transfected to U2OS cells (empty vs. ECS4; P=.011 by t test) (fig. 1B). This implies that the putative SHOX enhancer resides in ECS4 and interacts with the SHOX promoter on exon 2. Finally, we searched ECS4 for potential binding sites for transcription factors relevant to bone development, using the MATINSPECTOR, TESS, and TFSEARCH programs. The putative binding sites with the maximum core similarity of 1.0 and a matrix similarity >0.75 were identified for HOXA9, HOXB9, PBX1, and MEIS1, as well as for PBX1-HOXA9 and MEIS1-HOXA9 heterodimers, which are known to be involved in limb development (Mercader et al. 1999; Shanmugam et al. 1999; Zakany and Duboule 1999) (fig. 3). Thus, the sequence-specific DNA binding of such a factor(s) might mediate the enhancer activity for SHOX. However, other potential binding sites were also detected for various transcription factors, and the relevance to skeletal development has not been studied or excluded in most of the transcription factors. In addition, the binding sites remain unidentified for many transcription factors. Thus, further studies are necessary to define the enhancer sequence. Figure 3 Nucleotide sequence of ECS4 and putative binding sites for several transcription factors relevant to skeletal development. The human sequence is aligned with the chicken sequence. In summary, the results suggest that the ∼800-bp ECS4 harbors the putative downstream enhancer for SHOX transcription. This information will provide a useful clue for the clarification of the molecular network involved in SHOX-dependent skeletal development.

Backdoor pathway for dihydrotestosterone biosynthesis: Implications for normal and abnormal human sex development

We review the current knowledge about the “backdoor” pathway for the biosynthesis of dihydrotestosterone (DHT). While DHT is produced from cholesterol through the conventional “frontdoor” pathway via testosterone, recent studies have provided compelling evidence for the presence of an alternative “backdoor” pathway to DHT without testosterone intermediacy. This backdoor pathway is known to exist in the tammar wallaby pouch young testis and the immature mouse testis, and has been suggested to be present in the human as well. Indeed, molecular analysis has identified pathologic mutations of genes involved in the backdoor pathway in genetic male patients with undermasculinized external genitalia, and urine steroid profile analysis has argued for the relevance of the activated backdoor pathway to abnormal virilization in genetic females with cytochrome P450 oxidoreductase deficiency and 21‐hydroxylase deficiency. It is likely that the backdoor pathway is primarily operating in the fetal testis in a physiological condition to produce a sufficient amount of DHT for male sex development, and that the backdoor pathway is driven with a possible interaction between fetal and permanent adrenals in pathologic conditions with increased 17‐hydroxyprogesterone levels. These findings provide novel insights into androgen biosynthesis in both physiological and pathological conditions. Developmental Dynamics 242:320–329, 2013.

Monozygotic female twins discordant for Silver–Russell syndrome and hypomethylation of the H19-DMR

AbstractSilver-Russell syndrome (SRS) is characterized by growth failure and dysmorphic features, and is frequently caused by hypomethylation of the paternally derived H19-DMR (epimutation). We observed 5 8/12-year-old female twins discordant for SRS. One twin exhibited SRS-compatible features, such as pre- and postnatal growth failure, relative macrocephaly, triangular face, left hemihypotrophy, and bilateral fifth finger clinodactyly, whereas the other twin showed apparently normal phenotype. Microsatellite analysis for 26 loci on multiple chromosomes showed monozygosity. Methylation analysis for the H19-DMR indicated epimutation in roughly half of cells in the affected twin and normal patterns in the unaffected twin and the parents. X-inactivation analysis revealed random X-inactivation with a nearly identical pattern between the twins. The discordant methylation pattern of the H19-DMR may primarily be due to a failure to maintain the DNA methyltransferase-1-dependent methylation imprint around the pre-implantation S phase, because such failure would result in the production of two different cell clones, one with normally methylated DMR and the other with demethylated DMR, leading to the separation of cells with different characters and resultant twinning.