Long-read sequencing reveals the complex structure of extra dic(21;21) chromosome and its biological effects.

Complex congenital chromosome abnormalities are rare but often cause severe symptoms. However, the structures and biological impacts of such abnormalities have seldomly been analyzed at the molecular level. Previously, we reported a Japanese female patient with severe developmental defects. The patient had an extra dicentric chromosome 21 (chr21) consisting of two partial chr21 copies fused together within their long arms along with two centromeres and many copy number changes. In this study, we performed whole-genome, transcriptional, and DNA methylation analyses, coupled with novel bioinformatic approaches, to reveal the complex structure of the extra chromosome and its transcriptional and epigenetic changes. Long-read sequencing accurately identified the structures of junctions related to the copy number changes in extra chr21 and suggested the mechanism of the structural changes. Our transcriptome analysis showed the overexpression of genes in extra chr21. Additionally, an allele-specific DNA methylation analysis of the long-read sequencing data suggested that the centromeric region of extra chr21 was hypermethylated, a property associated with the inactivation of one centromere in the extra chromosome. Our comprehensive analysis provides insights into the molecular mechanism underlying the generation of the extra chromosome and its pathogenic roles.

Identification of a KDM6A somatic mutation responsible for Kabuki syndrome by excluding a conflicting KMT2D germline variant through episignature analysis.

Kabuki syndrome (KS) is a congenital disorder caused by mutations in either KMT2D on chromosome 12 or KDM6A on chromosome X, encoding a lysine methyltransferase and a lysine demethylase, respectively. A 9-year-4-month-old male patient with a normal karyotype presented with KS and autism spectrum disorder. Genetic testing for KS was conducted by Sanger sequencing and episignature analysis using DNA methylation array data. The patient had a mosaic stop-gain variant in KDM6A and a heterozygous missense variant (rs201078160) in KMT2D. The KDM6A variant is expected to be deleterious. The KMT2D variant pathogenicity has been inconsistently reported in the ClinVar database. Using biobanking resources, we identified two heterozygous individuals possessing the rs201078160 variant. In a subsequent episignature analysis, the KS patient showed the KS episignature, but two control individuals with the rs201078160 variant did not. Our results indicate that the mosaic stop-gained variant in KDM6A, but not the rs201078160 variant in KMT2D, is responsible for the KS phenotype in the patient. This study further demonstrated the utility of DNA methylation information in diagnosing rare genetic diseases and emphasized the importance of a reference dataset containing both genotype and DNA methylation information.

Adult acampomelic campomelic dysplasia and disorders of sex development due to a reciprocal translocation involving chromosome 17q24.3 upstream of the SOX9 gene.

Balanced chromosomal rearrangements with a breakpoint located upstream of the sex determining region Y-box 9 (SOX9) gene on chromosome 17q24.3 are associated with skeletal abnormalities, campomelic dysplasia (CMPD), or acampomelic campomelic dysplasia (ACMPD). We report on a female patient with a reciprocal translocation of t (11; 17) (p15.4; q24.3), who was diagnosed with acampomelic campomelic dysplasia. The 34-year-old Japanese patient presented with distinct skeletal abnormalities, profound intellectual disability, and female phenotype despite the presence of Y chromosome and the sex determining region Y (SRY) gene. Her menarche started at 33 years and 4 months after hormone therapy of estrogen therapy followed by estrogen progesterone therapy. By conducting whole genome sequencing followed by Sanger sequencing validation, we determined the precise breakpoint positions of the reciprocal translocation, one of which was located 203 kb upstream of the SOX9 gene. Considering the phenotypic variations previously reported among the CMPD/ACMPD patients with a chromosomal translocation in the vicinity of SOX9, the identified translocation was concluded to be responsible for all major phenotypes observed in the patient.

Tetrasomy 21 pter→q21.3 due to an extra +dic(21;21)mat in a severely psychomotor-retarded female patient without Down syndrome phenotype.

Complete or partial tetrasomy 21 has been reported only in rare cases. We report a Japanese female patient with tetrasomy 21 due to an extra chromosome derived from chromosome 21 (Chr21). The patient had severe psychomotor retardation without Down syndrome (DS) phenotype; she showed short stature, microcephaly, round face, cleft lip and palate, and other dysmorphic features. The chromosome analyses for the patient detected an extra dicentric Chr21 consisting of two partial Chr21 copies fused together within their long arms. Her karyotype was revealed to be 47,XX,+dic(21;21). Allelic ratios of heterozygous SNPs observed in the patient indicated the maternal origin of the extra Chr21. Copy number and structural variant analyses using whole genome sequencing data indicated that the distal breakpoint of the dicentric Chr21 was located within 21q21.3 and that the extra Chr21 did not simply consist of inverted duplications of the pter→q21.3 region, but likely contained multiple partial deletions, duplications, and inversions within it. Fluorescence in situ hybridization results were consistent with the karyotype and genomic analyses. The patient's lack of DS phenotype turned out to be due to the normal copy number of the DS critical region (21q22.13-22.3). A possible molecular mechanism leading to the complex genomic rearrangements in the tetrasomic region consists mainly of breakage-fusion-bridge cycles with an unequal crossing-over event.

Sex differences in congenital heart disease in Down syndrome: study data from medical records and questionnaires in a region of Japan.

Reports indicate lower Down syndrome (DS) survival among females than among males in Australia, contrasting with female longevity in the general population. Using data on 1310 people with DS (626 females and 684 males) in Japan from five hospitals' medical records and questionnaires completed by parents of people with DS, we investigated sex differences in congenital heart disease (CHD), which may be related to mortality. The CHD rate was significantly higher for females (354, 57%) than for males (338, 49%; p=0.010). Significantly more females (199, 32%) than males (175, 26%) underwent surgery for CHD (p=0.018).

Interstitial 22q13 deletions: genes other than SHANK3 have major effects on cognitive and language development.

The severe mental retardation and speech deficits associated with 22q13 terminal deletions have been attributed in large part to haploinsufficiency of SHANK3, which maps to all 22q13 terminal deletions, although more proximal genes are assumed to have minor effects. We report two children with interstitial deletions of 22q13 and two copies of SHANK3, but clinical features similar to the terminal 22q13 deletion syndrome, including mental retardation and severe speech delay. Both these interstitial deletions are completely contained within the largest terminal deletion, but do not overlap with the nine smallest terminal deletions. These interstitial deletions indicate that haploinsufficiency for 22q13 genes other than SHANK3 can have major effects on cognitive and language development. However, the relatively mild speech problems and normal cognitive abilities of a parent who transmitted her identical interstitial deletion to her more severely affected son suggests that the phenotype associated with this region may be more variable than terminal deletions and therefore contribute to the relative lack of correlation between clinical severity and size of terminal deletions. The phenotypic similarity between the interstitial deletions and non-overlapping small terminal 22q13 deletions emphasizes the general nonspecificity of the clinical picture of the 22q13 deletion syndrome and the importance of molecular analysis for diagnosis.

Associations of blood selenium and serum lipid levels in Japanese premenopausal and postmenopausal women.

OBJECTIVE: The purpose of this study was to investigate whether the level of selenium in serum or the level of selenium in erythrocytes (E-Se) was associated with serum lipid parameters (total cholesterol [TC], triglycerides, and high-density and low-density lipoprotein cholesterol [LDL-C]) in pre- and postmenopausal women. DESIGN: Healthy female workers aged 35 to 60 years were recruited, and TC, triglycerides, high-density lipoprotein cholesterol, LDL-C, E-Se, and serum-Se levels were measured in 68 premenopausal and 59 postmenopausal women after excluding those who were treated for hyperlipidemia, receiving hormone therapy, had previous gynecological surgeries, or had irregular menstrual cycles. Information on these women, such as smoking status, current status of menstruation, and current or previous medical treatments, was obtained from a self-administered questionnaire. Correlation analyses and stepwise multiple regression analysis were applied to reveal the interrelationship between serum lipids and Se levels after adjusting for age, body mass index, smoking, and menopause status. RESULTS: Postmenopausal women had significantly higher levels of TC, triglycerides, and LDL-C and lower levels of E-Se than premenopausal women. TC and LDL-C levels after menopause correlated negatively with E-Se. These associations were also statistically significant after controlling for age, smoking status, and body mass index. E-Se was selected as a significant related variable to explain TC and LDL-C levels as well as body mass index by multiple regression analysis. CONCLUSION: E-Se may be one of the possible associated factors that decrease the levels of TC and LDL-C after menopause. Further studies are needed to clarify the cause-and-effect relationship of nutritional Se status and lipid profile.

Variations in plasma selenium levels as a result of the menstrual cycle and pregnancy in healthy Japanese women.

Plasma levels of selenium (Se) were determined consecutively during a menstrual cycle of six women in three phases (i.e., menses, follicular, and luteal). To detect possible differences in relation to normal pregnancy, plasma levels of Se were also determined in paired samples of maternal and umbilical cord blood from 12 pregnant women. No periodic changes in the plasma Se levels were observed during the menstrual cycle. The intraindividual variation, estimated by coefficients of variation, ranged from 1.9% to 9.9% among the menstrual phases of the subjects. The plasma Se level during pregnancy did not differ significantly from those of nonpregnant women, and those in the second trimester and at delivery were at similar levels (1.58+/-.14 and 1.48+/-.20 mmol/L, respectively). Compared to the levels of maternal Se at delivery, the fetal cord plasma at birth had a significant lower Se level (1.23+/-.34 mmol/L, p<.05).

True hermaphroditism with 46,X,+22p/46,XY and gonadal mosaicism detected by fluorescence in situ hybridization.

A Japanese girl was diagnosed as true hermaphroditism with 46,X,+mar/46,XY and the marker chromosome was determined on the short arm of chromosome 22 without alpha-satellite by fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) methods. At birth, she showed intersexual external genitalia, urethral-vaginal fistula and right inguinal hernia. The right gonad was revealed as an ovotestis, and the left was as an undifferentiated testis. The gonadal mosaicism was demonstrated directly in gonadal tissue by interphase FISH.

Epithelia-mesenchyme interaction plays an essential role in transdifferentiation of retinal pigment epithelium of silver mutant quail: localization of FGF and related molecules and aberrant migration pattern of neural crest cells during eye rudiment formation.

Homozygotes of the quail silver mutation, which have plumage color changes, also display a unique phenotype in the eye: during early embryonic development, the retinal pigment epithelium (RPE) spontaneously transdifferentiates into neural retinal tissue. Mitf is considered to be the responsible gene and to function similarly to the mouse microphthalmia mutation, and tissue interaction between RPE and surrounding mesenchymal tissue in organ culture has been shown to be essential for the initiation of the transdifferentiation process in which fibroblast growth factor (FGF) signaling is involved. The immunohistochemical results of the present study show that laminin and heparan sulfate proteoglycan, both acting as cofactors for FGF binding, are localized in the area of transdifferentiation of silver embryos much more abundantly than in wild-type embryos. More intense immunohistochemical staining with FGF-1 antibody, but not with FGF-2 antibody, is also found in the neural retina, RPE, and choroidal tissue of silver embryos than in wild-type embryos. HNK-1 immunohistochemistry revealed that clusters of HNK-1-positive cells (presumptive migrating neural crest cells) are frequently located around the developing eyes and in the posterior region of the silver embryonic eye. Finally, chick-quail chimerical eyes were made by grafting silver quail optic vesicles to chicken host embryos: in most cases, no transdifferentiation occurs in the silver RPE, but in a few cases, transdifferentiation occurs where silver quail cells predominate in the choroid tissue. These observations together with our previous in vitro study indicate that the silver mutation affects not only RPE cells but also cephalic neural crest cells, which migrate to the eye rudiment, and that these crest cells play an essential role in the transdifferentiation of RPE, possibly by modifying the FGF signaling pathway. The precise molecular mechanism involved in RPE-neural crest cell interaction is still unknown, and the quail silver mutation is considered to be a good experimental model for studying the role of neural crest cells in vertebrate eye development.