Delineation of clinical features in Wiedemann-Steiner syndrome caused by KMT2A mutations.

Wiedemann-Steiner syndrome (WSS) is an autosomal dominant congenital anomaly syndrome characterized by hairy elbows, dysmorphic facial appearances (hypertelorism, thick eyebrows, downslanted and vertically narrow palpebral fissures), pre- and post-natal growth deficiency, and psychomotor delay. WSS is caused by heterozygous mutations in KMT2A (also known as MLL), a gene encoding a histone methyltransferase. Here, we identify six novel KMT2A mutations in six WSS patients, with four mutations occurring de novo. Interestingly, some of the patients were initially diagnosed with atypical Kabuki syndrome, which is caused by mutations in KMT2D or KDM6A, genes also involved in histone methylation. KMT2A mutations and clinical features are summarized in our six patients together with eight previously reported patients. Furthermore, clinical comparison of the two syndromes is discussed in detail.

Down-regulation of ABCC11 protein (MRP8) in human breast cancer.

AIM: To investigate the expression of ABCC11 (MRP8) protein in normal breast tissue, and examine the difference in ABCC11 mRNA and protein expression between normal breast and breast cancer tissues taking into account ABCC11 genotype (a functional SNP, rs17822931) and estrogen receptor (ER) status. METHODS: Sections of paraffin-embedded normal and malignant tissues from 10 patients with invasive ductal carcinoma were used for immunohistochemical analysis. DNA and RNA were extracted from the same sections and used for genotyping and ABCC11 transcript expression measurement by quantitative RT-PCR. RESULTS: A strong expression of ABCC11 was found in epithelial and myoepithelial cells of normal breast lobules and ducts in individuals with different ABCC11 genotypes. A predominant decrease of ABCC11 expression was observed in malignant tissue compared to normal beast specimen (8 of 10 cases), despite four out of ten tumors showed the elevated ABCC11 mRNA level as compared to the normal counterpart. Neither ABCC11 mRNA nor protein expression in normal or cancerous tissue correlated with ER status. CONCLUSION: The expression of ABCC11 protein appears to be decreased in most BC. The effect of ABCC11 protein on breast cancer chemosensitivity is likely to be more complex than that which can be directly inferred from ABCC11 genotype and mRNA expression level in the tumor.

A novel missense mutation in Van der Woude syndrome: usefulness of fingernail DNA for genetic analysis.

Van der Woude syndrome (VWS) is an autosomal-dominant oral facial disorder. To find a gene mutation in a Japanese family using fingernail DNA samples, we performed this study. We hypothesized that a gene mutation in IRF6 might be involved in VWS, and that fingernail DNA samples may be valuable for detecting such mutations. Linkage and haplotype analyses of the family mapped the disease locus to the 1q32-q41 region. Mutation analysis with an improved extraction method for fingernail DNA detected a novel missense mutation (1046A>T, E349V) in exon 7 of IRF6 in all the affected members of the family. Since the E349V change may disturb the hydrophobic core and affect regulatory activity of IRF6, it is most likely that the mutation is causative for VWS in this family. Fingernail DNA is thus useful for linkage and mutation analyses, since the fingernail can be easily obtained non-invasively, sent through the mail, and stored for a long period. We emphasize here the usefulness of fingernail DNA for the genetic analysis of a disease.

Patched homologue 1 mutations in four Japanese families with basal cell nevus syndrome.

AIM: To search for patched homologue 1 (PTCH1) mutations in four families with basal cell nevus syndrome (BCNS). METHODS: Mutation analysis of PTCH1 in unrelated Japanese families affected with BCNS was carried out by direct sequencing. RESULTS: Six novel PTCH1 mutations, 833G-->A in exon 6, 1415C-->A and 1451G-->T in exon 10, 2798delC in exon 17, 2918-2925dupAGTTCCCT in exon 18 and 3956C-->A in exon 23, were identified. CONCLUSIONS: Among the six PTCH1 mutations, two frameshift mutations (2798delC and 2918-2925dupAGTTCCCT) and one nonsense mutation (833G-->A) are predicted to lead to premature termination of PTCH1 protein translation. Three simultaneous mutations, 1415C-->A (A472D) and 1451G-->T (G484V) in exon 10, and 3956G-->A (R1319H) in exon 23, were found on one allele in only affected members in one family and none of them were found among 90 unrelated healthy Japanese. The three mutations on one chromosome may have resulted from errors in the recombinational repair process and this is the first report on the PTCH1 mutations due to such a mechanism.

Growth hormone therapy and scoliosis in patients with Prader-Willi syndrome.

Growth hormone (GH) therapy for short stature in patients with Prader-Willi syndrome (PWS) has started worldwide, and various favorable effects have been reported. However, the possibility of progression of scoliosis arises as a new problem of the GH therapy. In this study, we analyzed whether 72 patients who have been followed up in our hospital have such a problem. They included 46 males and 26 females (41 patients with the GH therapy and 31 without it) aged from one to 49 years. Consequently, 33 (45.8%) of 72 patients had scoliosis with the Cobb angle of >10 degrees. Twenty (48.8%) of forty-one patients who received a GH therapy and 13 (41.9%) of 31 patients without the therapy had scoliosis, the frequency of scoliosis between the two groups showing no statistical difference (P = 0.56). Height velocity of scoliotic and non-scoliotic patients during the first year of the therapy was 8.59 +/- 1.92 and 10.70 +/- 2.54 cm, respectively, showing a significant difference (P < 0.001). This shows that accelerated height velocity may not induce scoliosis. Comparison of starting age of a GH treatment revealed that non-scoliotic patients received the therapy earlier than scoliotic patients (P = 0.021). Among 20 scoliotic patients who received the GH therapy, the degree of scoliosis progressed during the therapy in six patients, improved in three and fluctuated in one. Many patients showed progression of scoliosis with age irrespective of the use of GH, and some patients improved their scoliosis during the GH therapy. These findings showed that a GH therapy increases height velocity of PWS patients but does not necessarily develop scoliosis, and early start of the therapy may not be an exacerbating factor of scoliosis.

Non-hotspot-related breakpoints of common deletions in Sotos syndrome are located within destabilised DNA regions.

BACKGROUND: Sotos syndrome (SoS) is a disorder characterised by excessive growth, typical craniofacial features, and developmental retardation. It is caused by haploinsuffiency of NSD1 at 5q35. There is a 3.0 kb recombination hotspot in which the breakpoints of around 80% of SoS patients with a common deletion can be mapped. OBJECTIVE: To identify deletion breakpoints located outside the SoS recombination hotspot. METHODS: A screening system for the directly orientated segments of the SoS LCRs was developed for 10 SoS patients with a common deletion who were negative for the SoS hotspot. Deletion-junction fragments were analysed for DNA duplex stability and their relation to scaffold/matrix attachment regions (S/MARs). These features were compared with the SoS hotspot and recombination hotspots of other genomic disorders. RESULTS: The breakpoint was mapped in four SoS patients, two with a deletion in the maternally derived chromosome. These breakpoint regions were located approximately 2.5 kb, approximately 9.6 kb, approximately 27.2, and approximately 27.7 kb telomeric to the SoS hotspot and were confined to 164 bp, 46 bp, 256 bp, and 124 bp, respectively. Two of the regions were mapped within Alu elements. All crossover events were found to have occurred within or adjacent to a highly destabilised DNA duplex with a high S/MAR probability. In contrast, the SoS hotspot and other genomic disorders' recombination hotspots were mapped to stabilised DNA helix regions, flanked by destabilised regions with high probability of containing S/MAR elements. CONCLUSIONS: The data suggest that a specific chromatin structure may increase susceptibility for recurrent crossover events and thus predispose to recombination hotspots in genomic disorders.

Cause of sudden, unexpected death of Prader-Willi syndrome patients with or without growth hormone treatment.

Patients with Prader-Willi syndrome (PWS) are recognized to have a tendency of sudden, unexpected death (SED), but its exact cause is unknown because of paucity of such case reports. Since growth hormone (GH) treatment was applied to PWS patients worldwide, several cases of death have been reported. However, whether the therapy is directly related to their SED remains unknown, too. We collected 13 deceased PWS patients (Group A, aged 9 months to 34 years) who had never received GH therapy, and seven deceased patients (Group B, all boys aged 0.7-15 years) having received the therapy from the registration in PWS-patient-support associations and from the literature, respectively. We then compared the cause of SED between the two groups. Irrespective of GH therapy, SED of infants under age 1 year was associated with milk aspiration or hypothalamic dysregulation of respiration, while SED of patients in early childhood or adolescence occurred at sleeping in association with preceding viral infections. In contrast, SED of four adult (>20 years of age) patients who never received GH therapy was associated with complications, such as leg cellulites and pulmonary embolism, secondary to massive obesity and diabetes mellitus (DM). Two Group-B patients (aged 14 and 20 years) without any obesity-related or diabetes-related complications died of drowning in a bath tub, and their drowning death could be related to poor respiratory control. These findings indicated that the cause of SED is not essentially different between PWS patients with and without GH treatment. Deceased PWS patients may have had underlying respiratory dysregulation and hypothalamic dysfunction, and GH therapy might have led to certain obstructive respiratory disturbances that exacerbated the respiratory conditions. This will call clinicians' attention when using GH in PWS patients, for example, careful determination of the dose of GH and careful monitoring of patient's respiratory conditions, especially in male obese patients with respiratory problems.

Neurons but not glial cells show reciprocal imprinting of sense and antisense transcripts of Ube3a.

The human UBE3A gene shows brain-specific partial imprinting, and lack of a maternally inherited allele causes Angelman syndrome (AS), which is characterized by neurobehavioral anomalies. In several AS model mice, imprinted Ube3a expression is detected predominantly in the hippocampus, cerebellar Purkinje cells and the olfactory bulb. Therefore, imprinting of mouse Ube3a is thought to be region-specific with different levels of silencing of the paternal Ube3a allele in different brain regions. To determine cell types of imprinted Ube3a expression, we analyzed its imprinting status in embryonic brain cells by using primary cortical cell cultures. RT-PCR and immunofluorescence were performed to determine the allelic expression of the gene. The Ube3a gene encodes two RNA transcripts in the brain, sense and antisense. The sense transcript was expressed maternally in neurons but biallelically in glial cells in the embryonic brain, whereas the antisense transcript was expressed only in neurons and only from the paternal allele. Our data present evidence of brain cell type-specific imprinting, i.e. neuron-specific imprinting of Ube3a in primary brain cell cultures. Reciprocal imprinting of sense and antisense transcripts present only in neurons suggests that the neuron-specific imprinting mechanism is related to the lineage determination of neural stem cells.

PA26 is a candidate gene for heterotaxia in humans: identification of a novel PA26-related gene family in human and mouse.

Heterotaxia is an aetiologically heterogeneous condition caused by an abnormal left-right axis formation, resulting in reversed left-right polarity of one or more organ systems. In a patient with heterotaxia and a de novo reciprocal translocation t(6;18)(q21;q21), we found that the PA26 gene was disrupted by the 6q21 breakpoint. Northern blot analysis showed decreased expression of the PA26 gene in an Epstein-Barr virus-transformed cell line of this patient. During early embryogenesis of Xenopus, the orthologue of PA26, XPA26 is exclusively expressed in the notochord, a midline structure. This further supports a possible role of PA26 in human situs determination. Mutation analysis of human PA26 gene in 40 unrelated individuals with unexplained heterotaxia failed to identify mutations, indicating that PA26 mutations are not a frequent cause of heterotaxia in humans. Analysis of the PA26 gene structure resulted in the identification of a novel PA26-related gene family, which we have named the sestrin family, and which comprises three closely related genes in human and in mouse.

Molecular characterization of NSD1, a human homologue of the mouse Nsd1 gene.

NR-binding SET-domain-containing protein (NSD1) is a mouse nuclear protein containing su(var)3-9, enhancer-of-zeste, trithorax (SET), proline-tryptophan-tryptophan-proline (PWWP) and plant homeodomain protein (PHD)-finger domains (Huang et al., EMBO J. 17 (1998) 3398). This protein also has two other distinct nuclear receptor (NR)-interaction domains, called NID(-L) and NID(+L), and acts as both a NR corepressor and a coactivator by interacting directly with the ligand-binding domain of several NRs. Thus, NSD1 is a bifunctional, transcriptional, intermediary factor. We isolated the human homologue (NSD1) of the mouse NSD1 gene (Nsd1), mapped it to human chromosome 5q35, and characterized its genomic structure. NSD1 consists of at least 23 exons. Its cDNA is 8552 bp long, has an 8088 bp open reading frame, contains at least six functional domains (SET, PWWP-I, PWWP-II, PHD-I, PHD-II, and PHD-III) and ten putative nuclear localization signals, and encodes 2696 amino acids. NSD1 shows 86% identity with the mouse Nsd1 at the nucleotide level, and 83% at the amino acid level. NSD1 is expressed in the fetal/adult brain, kidney, skeletal muscle, spleen, and the thymus, and faintly in the lung. Two different transcripts (9.0 and 10.0 kb) were consistently observed in various fetal and adult tissues examined. These findings favor the character of NSD1 as a nucleus-localized, basic transcriptional factor and also a bifunctional transcriptional regulator, such as that of the mouse Nsd1. It remains to be investigated whether mutations of NSD1 lead to a specific phenotype in man.