Hiroshi Yoshihashi

Imprinting of human GRB10 and its mutations in two patients with Russell-Silver syndrome.

Documentation of maternal uniparental disomy of chromosome 7 in 10% of patients with Russell-Silver syndrome (RSS), characterized by prenatal and postnatal growth retardation and dysmorphic features, has suggested the presence of an imprinted gene on chromosome 7 whose mutation is responsible for the RSS phenotype. Human GRB10 on chromosome 7, a homologue of the mouse imprinted gene Grb10, is a candidate, because GRB10 has a suppressive effect on growth, through its interaction with either the IGF-I receptor or the GH receptor, and two patients with RSS were shown to have a maternally derived duplication of 7p11-p13, encompassing GRB10. In the present study, we first demonstrated that the GRB10 gene is also monoallelically expressed in human fetal brain tissues and is transcribed from the maternally derived allele in somatic-cell hybrids. Hence, human GRB10 is imprinted. A mutation analysis of GRB10 in 58 unrelated patients with RSS identified, within the N-terminal domain of the protein, a P95S substitution in two patients with RSS. In these two cases, the mutant allele was inherited from the mother. The fact that monoallelic GRB10 expression was observed from the maternal allele in this study suggests but does not prove that these maternally transmitted mutant alleles contribute to the RSS phenotype.

Further delineation of 9q22 deletion syndrome associated with basal cell nevus (Gorlin) syndrome : Report of two cases and review of the literature

Basal cell nevus syndrome (BCNS; Gorlin syndrome) is an autosomal dominant disorder, characterized by a predisposition to neoplasms and developmental abnormalities. BCNS is caused by mutations in the human homolog of the Drosophila patched gene‐1, PTCH1, which is mapped on chromosome 9q22.3. Nonsense, frameshift, in‐frame deletions, splice‐site, and missense mutations have been found in the syndrome. Haploinsufficiency of PTCH1, which is caused by interstitial deletion of 9q22.3, is also responsible for the syndrome. To date, 19 cases with interstitial deletion of long arm of chromosome 9 involving the region of q22 have been reported. We describe two unrelated patients with some typical features of BCNS associated with deletion of 9q21.33‐q31.1 and determined the boundary of the deletion by fluorescence in situ hybridization (FISH) with bacterial artificial chromosome (BAC) clones. The results showed that the size of deletions is between 15.33 and 16.04 Mb in patient 1 and between 18.08 and 18.54 Mb in patient 2. Although the size and breakpoints were different from those of previously reported cases, the clinical features are common to patients with 9q22 deletion associated with BCNS. Delineation of the 9q22 deletions and further consideration of the genes responsible for the characteristic manifestations may provide insight into this newly recognized deletion syndrome.

1p36 deletion syndrome associated with Prader-Willi-like phenotype.

Background:  1p36 deletion syndrome is one of the most common subtelomeric deletion syndromes, characterized by moderate to severe mental retardation, characteristic facial appearance, hypotonia, obesity, and seizures. The clinical features often overlap with those of Prader–Willi syndrome (PWS). To elucidate the phenotype–genotype correlation in 1p36 deletion syndrome, two cases involving a PWS‐like phenotype were analyzed on molecular cytogenetics.

Clinical application of array-based comparative genomic hybridization by two-stage screening for 536 patients with mental retardation and multiple congenital anomalies

Recent advances in the analysis of patients with congenital abnormalities using array-based comparative genome hybridization (aCGH) have uncovered two types of genomic copy-number variants (CNVs); pathogenic CNVs (pCNVs) relevant to congenital disorders and benign CNVs observed also in healthy populations, complicating the screening of disease-associated alterations by aCGH. To apply the aCGH technique to the diagnosis as well as investigation of multiple congenital anomalies and mental retardation (MCA/MR), we constructed a consortium with 23 medical institutes and hospitals in Japan, and recruited 536 patients with clinically uncharacterized MCA/MR, whose karyotypes were normal according to conventional cytogenetics, for two-stage screening using two types of bacterial artificial chromosome-based microarray. The first screening using a targeted array detected pCNV in 54 of 536 cases (10.1%), whereas the second screening of the 349 cases negative in the first screening using a genome-wide high-density array at intervals of approximately 0.7 Mb detected pCNVs in 48 cases (13.8%), including pCNVs relevant to recently established microdeletion or microduplication syndromes, CNVs containing pathogenic genes and recurrent CNVs containing the same region among different patients. The results show the efficient application of aCGH in the clinical setting.

A de novo mutation (R279C) in the P63 gene in a patient with EEC syndrome

To the Editor: Ectrodactyly–ectodermal dysplasia–clefting (EEC) syndrome is an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia and cleft lip with or without cleft palate (1). Celli et al. demonstrated that heterozygous mutations in the P63 gene, a cell cycle regulator on chromosome 3q27, lead to the EEC syndrome (2). To date, a total of twelve heterozygous nucleotide changes in P63 has been detected in EEC syndrome patients (2–4). We report here the first Asian EEC syndrome patient with a P63 mutation. The patient was delivered at 40 weeks gestation to a 22-year-old G1P0 woman. The birth weight of 2820 g was in the 25th percentile, and the patient’s length of 49 cm was in the 50th percentile. The patient’s head circumference of 33 cm was in the 25th percentile. At birth, the diagnosis of the EEC syndrome was made based on middle ray defects of both hands and feet (Fig. 1), sparse scalp, eyebrow, and eyelash hair, thin and dry skin, and cleft lip with cleft palate. After obtaining informed consent from the parents, the coding sequence of P63 was screened for mutations by polymerase chain reaction (PCR) sequencing with primers designed by Celli et al. (2). Genomic DNA was extracted from whole blood with a QIAamp DNA Blood Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Sequencing of the PCR product amplified from exon 7 and flanking introns of P63 with primer 7F (5 -GGGAAG AACTGAGAAGGAACA AC-3 ) and primer 7R (5 -CAGCCACGATTTCACTTTGCC-3 ) revealed a heterozygous C to T transition at nucleotide 980 (with respect to the adenine of the start codon) (Fig. 2). This mutation predicts an arginine-to-cysteine substitution at amino acid 279 within the DNA binding domain (numbering based on the TA-P63 isotype). The C to T transition was not present in either parent or 200 ethnically matched control chromosomes. No other potentially pathogenic P63 sequence variations were identified. We conclude that the R279C mutation is pathogenic for the following reasons: 1) The R279C substitution occurred within the DNA-binding domain showing an extreme degree of evolutionary sequence conservation among vertebrates. Furthermore, protein sequences flanking the mutation are strictly conserved among three related proteins: P53, P63, and P73. 2) A crystallographic study of the P53 protein predicts the arginine residue at 248, which corresponds to the arginine 279 of P63, to extend into the minor groove of the DNA (5). 3) The R279C substitution was not identified in either parent or normal controls. The arginine residue at 279 was mutated to histidine in 2 patients with the EEC syndrome (2, 3). Hence, the arginine residue at 279 represents a mutational hotspot. Amino acid substitutions in the P63 protein sequence involving the sterile alpha motif (SAM) domain, as opposed to the DNA-binding domain, lead to the Hay–Wells syndrome (OMIM 106260) characterized by ankyloblephalon (fused eyelids) and severe scalp dermatitis as distinguishing features (6). Twelve patients with the Hay–Wells

Large fontanelles are a shared feature of haploinsufficiency of RUNX2 and its co-activator CBFB.

ABSTRACT CBFB at 16q22 heterodimerizes with either RUNX2 (also known as CBFA1) or RUNX1 (CBFA2) to activate the transcription of downstream molecules. RUNX2 regulates osteoblast differentiation and chondrocyte maturation and its haploinsufficiency leads to cleidocranial dysplasia, characterized large fontanelles, hypoplasia or aplasia of the clavicles, hypoplasia of the distal phalanges, and a wide pubic symphysis. Complete loss of Runx1 or Cbfb in mice is lethal because of the absence of fetal liver hematopoiesis. Fetal rescue in Cbfb–/– mice by providing the Cbfb functions in the hematopoietic progenitors leads to wide fontanelle and delayed chondrocyte maturation, presumably resulting from the incomplete function of the transcriptional pathway mediated by the Cbfb‐Runx2 heterodimer. The present report describes a patient with a small deletion of chromosome 16q22.1 encompassing CBFB. Skeletal abnormalities included a widely open fontanelle, multiple wormian bones along the sagittal suture, hypoplasia of the distal phalanges, and mildly shortened clavicles. G‐banding analysis revealed a shortening of the 16q22.1 band. A fluorescence in situ hybridization analysis, using the BAC probe spanning the CBFB locus at 16q22.1, revealed that the CBFB probe hybridized to only one of the two homologous chromosome 16 regions. Array‐comparative genomic hybridization analysis revealed that the deletion spans 1.2 megabases. In reviewing eight previously reported cases of 16q interstitial deletions involving band q22, large cranial sutures were noted in all but one case. Considering the phenotypic similarity of the 16q22 deletion case and Cbfb–/– mice rescued for hematopoiesis and the consistency of the phenotype among 16q22 deletion cases, we suggest that the common phenotypic feature of the 16q22 deletion, large fontanelles, can be attributed to a haploinsufficiency of CBFB.

Human homolog of the mouse imprinted gene Impact resides at the pericentric region of chromosome 18 within the critical region for bipolar affective disorder

Several mapping studies of families with multiple individuals who have bipolar affective disorder (BPAD) have demonstrated possible linkage of the trait to the pericentric region of chromosome 18 (18cen). Currently, the large size of the critical interval defined by these studies makes effective selection of candidate genes formidable. However, documentation of 18cen-linked families in which a parent-of-origin effect was observed in the transmission of the BPAD trait provides a clue to the nature of the putative gene; it may be imprinted. In the present study, we cloned IMPACT, the human homolog of the mouse imprinted gene Impact and mapped it to 18cen within the critical interval for BPAD. Human IMPACT encodes a protein with 320 amino acids and is expressed at high levels in the brain. Since only a small number of imprinted genes are estimated to be present in the entire genome, very few imprinted genes would be expected to be present in this particular chromosomal region. Hence, IMPACT represents a candidate gene for BPAD susceptibility. Alternatively, other as yet unknown imprinted gene(s) adjacent to IMPACT could contribute to the BPAD trait, since multiple imprinted genes may occasionally form clusters. Localization of human IMPACT at 18cen in this study defines a promising target region in which to search for putative BPAD genes.

Japanese founder duplications/triplications involving BHLHA9 are associated with split-hand/foot malformation with or without long bone deficiency and Gollop-Wolfgang complex

BackgroundLimb malformations are rare disorders with high genetic heterogeneity. Although multiple genes/loci have been identified in limb malformations, underlying genetic factors still remain to be determined in most patients.MethodsThis study consisted of 51 Japanese families with split-hand/foot malformation (SHFM), SHFM with long bone deficiency (SHFLD) usually affecting the tibia, or Gollop-Wolfgang complex (GWC) characterized by SHFM and femoral bifurcation. Genetic studies included genomewide array comparative genomic hybridization and exome sequencing, together with standard molecular analyses.ResultsWe identified duplications/triplications of a 210,050 bp segment containing BHLHA9 in 29 SHFM patients, 11 SHFLD patients, two GWC patients, and 22 clinically normal relatives from 27 of the 51 families examined, as well as in 2 of 1,000 Japanese controls. Families with SHFLD- and/or GWC-positive patients were more frequent in triplications than in duplications. The fusion point was identical in all the duplications/triplications and was associated with a 4 bp microhomology. There was no sequence homology around the two breakpoints, whereas rearrangement-associated motifs were abundant around one breakpoint. The rs3951819-D17S1174 haplotype patterns were variable on the duplicated/triplicated segments. No discernible genetic alteration specific to patients was detected within or around BHLHA9, in the known causative SHFM genes, or in the exome.ConclusionsThese results indicate that BHLHA9 overdosage constitutes the most frequent susceptibility factor, with a dosage effect, for a range of limb malformations at least in Japan. Notably, this is the first study revealing the underlying genetic factor for the development of GWC, and demonstrating the presence of triplications involving BHLHA9. It is inferred that a Japanese founder duplication was generated through a replication-based mechanism and underwent subsequent triplication and haplotype modification through recombination-based mechanisms, and that the duplications/triplications with various haplotypes were widely spread in Japan primarily via clinically normal carriers and identified via manifesting patients. Furthermore, genotype-phenotype analyses of patients reported in this study and the previous studies imply that clinical variability is ascribed to multiple factors including the size of duplications/triplications as a critical factor.

Microduplication of Xq24 and Hartsfield syndrome with holoprosencephaly, ectrodactyly, and clefting.

The combination of holoprosencephaly and ectrodactyly, also known as Hartsfield syndrome, represents a unique genetic entity. An X‐linked recessive mode of transmission has been suggested for this condition based on the observation that male patients have preferentially been affected. Thus far, no candidate genes have been suggested on the X chromosome. We report a male patient with a full‐blown Hartsfield syndrome phenotype who had microduplication at Xq24 involving four genes. He presented with bilateral ectrodactyly of the hands (both hands had four fingers with a deep gap between the 2nd and 3rd digits), cleft lip and palate, and a depressed nasal bridge. Magnetic resonance imaging of the brain revealed lobar holoprosencephaly. His G‐banded karyotype was normal. Array comparative genomic hybridization (CGH) using the Agilent 244K Whole Human Genome CGH array revealed a microduplication at Xq24 of 210 kb. Parental testing revealed that the deletion was derived from the asymptomatic mother. Of the genes on the duplicated interval, the duplications of SLC25A43 and SLC25A5 appeared to be the most likely to explain the patients phenotype. From a clinical standpoint, it is important to point out that the propositus, who performs relatively well with holoprosencephaly and has a developmental quotient around 70, has survived multiple life‐threatening episodes of hypernatremia. Awareness of the risk of hypernatremia is of great importance for the anticipatory management of patients with ectrodactyly and an oral cleft, even in the absence of overt hypotelorism.

The incidence of hypoplasia of the corpus callosum in patients with dup (X)(q28) involving MECP2 is associated with the location of distal breakpoints.

Duplications of Xq28 harboring the methyl CpG binding protein 2 (MECP2) gene explain approximately 1% of X‐linked intellectual disability (XLID). The common clinical features observed in patients with dup(X)(q28) are severe ID, infantile hypotonia, mild dysmorphic features and a history of recurrent infections, and MECP2 duplication syndrome is now recognized as a clinical entity. While some patients with this syndrome have other characteristic phenotypes, the reason for the spectrum of phenotypes has not been clarified. Since dup(X)(q28) rearrangements vary in size and location, genes other than MECP2 might affect the phenotype. We used a high‐density oligonucleotide array to carry out precise mapping in eight Japanese families in which dup(X)(q28) was detected using an in‐house bacterial artificial chromosome‐based microarray to screen cohorts of individuals with multiple congenital anomalies and intellectual disability (MCA/ID) or with XLID. We hypothesized that the size, gene content, and location of dup(X)(q28) may contribute to variable expressively observed in MECP2 duplication syndrome. Genotype–phenotype correlation in our cases together with cases reported in the literature suggested that copy‐number gains between two low copy repeats (LCRK1 and LCRL1) are associated with the incidence of hypoplasia of the corpus callosum. Further studies are necessary to understand the mechanism of this association.