Genomic Characterization of Chromosomal Insertions: Insights into the Mechanisms Underlying Chromothripsis.

Chromosomal insertions are rare structural rearrangements, and the molecular mechanisms underlying their origin are unknown. In this study, we used whole genome sequencing to analyze breakpoints and junction sequences in 4 patients with chromosomal insertions. Our analysis revealed that none of the 4 cases involved a simple insertion mediated by a 3-chromosomal breakage and rejoining events. The inserted fragments consisted of multiple pieces derived from a localized genomic region, which were shuffled and rejoined in a disorderly fashion with variable copy number alterations. The junctions were blunt ended or with short microhomologies or short microinsertions, suggesting the involvement of nonhomologous end-joining. In one case, analysis of the parental origin of the chromosomes using nucleotide variations within the insertion revealed that maternal chromosomal segments were inserted into the paternal chromosome. This patient also carried both maternal alleles, suggesting the presence of zygotic trisomy. These data indicate that chromosomal shattering may occur in association with trisomy rescue in the early postzygotic stage.

The spectrum of ZEB2 mutations causing the Mowat-Wilson syndrome in Japanese populations.

Mowat-Wilson syndrome (MWS) is a multiple congenital anomaly syndrome characterized by moderate or severe intellectual disability, a characteristic facial appearance, microcephaly, epilepsy, agenesis or hypoplasia of the corpus callosum, congenital heart defects, Hirschsprung disease, and urogenital/renal anomalies. It is caused by de novo heterozygous loss of function mutations including nonsense mutations, frameshift mutations, and deletions in ZEB2 at 2q22. ZEB2 encodes the zinc finger E-box binding homeobox 2 protein consisting of 1,214 amino acids. Herein, we report 13 nonsense and 27 frameshift mutations from 40 newly identified MWS patients in Japan. Although the clinical findings of all the Japanese MWS patients with nonsense and frameshift mutations were quite similar to the previous review reports of MWS caused by nonsense mutations, frameshift mutations and deletions of ZEB2, the frequencies of microcephaly, Hirschsprung disease, and urogenital/renal anomalies were small. Patients harbored mutations spanning the region between the amino acids 55 and 1,204 in wild-type ZEB2. There was no obvious genotype-phenotype correlation among the patients. A transfection study demonstrated that the cellular level of the longest form of the mutant ZEB2 protein harboring the p.D1204Rfs*29 mutation was remarkably low. The results showed that the 3'-end frameshift mutation of ZEB2 causes MWS due to ZEB2 instability.

Mutation and functional analysis of ABCC2/multidrug resistance protein 2 in a Japanese patient with Dubin-Johnson syndrome.

Dubin-Johnson syndrome (DJS) is a recessive inherited disorder characterized by conjugated hyperbilirubinemia. It is caused by dysfunction of adenosine triphosphate-binding cassette, sub-family C, member 2 (ABCC2/MRP2) on the canalicular membrane of hepatocytes. We performed mutational analysis of the ABCC2/MRP2 gene in a Japanese female with DJS. Furthermore, we investigated the effects of the two identified DJS-associated mutations on MRP2 function. We found a compound heterozygous mutation in the patient: W709R (c.2124T>C), a missense mutation in exon 17, and R1310X (c.3928C>T), a nonsense mutation in exon 28. DJS-associated mutations have been shown to impair the protein maturation and transport activity of ABCC2/MRP2. We established HEK293 cell lines stably expressing one of the two identified DJS-associated mutations. Expressed W709R MRP2 was mainly core-glycosylated, predominantly retained in the endoplasmic reticulum, and exhibited no transport activity, suggesting that this mutation causes deficient maturation and impaired protein sorting. No MRP2 protein was expressed from HEK293 cells transfected with an R1310X-containing construct. This compound heterozygous mutation of the MRP2 gene causes dysfunction of the MRP2 protein and the hyperbilirubinemia seen in DJS.

Gas chromatographic-mass spectrometric determination of erythrocyte 3-deoxyglucosone in diabetic patients.

To determine if the erythrocyte levels of 3-deoxyglucosone (3-DG) are increased in diabetic patients, and if they correlate with glycemic status, they were measured in diabetic patients without renal disease as well as in healthy subjects. The erythrocyte levels of 3-DG were measured by a selected ion monitoring method of gas chromatography-chemical ionization mass spectrometry using [(13)C(6)]-3-DG as an internal standard. The erythrocyte levels of 3-DG were significantly higher in diabetic patients than in healthy subjects. The erythrocyte concentration of 3-DG was significantly and positively correlated with HbA1c (r=0.84, P<0.001). However, no significant correlation could be found between erythrocyte 3-DG and age, onset age of diabetes, or duration of diabetes in our group of diabetic patients. In diabetes, the production of 3-DG in the erythrocytes is increased via the polyol pathway and/or the Maillard reaction due to hyperglycemia.

Mutation analysis in a patient with succinic semialdehyde dehydrogenase deficiency: a compound heterozygote with 103-121del and 1460T > A of the ALDH5A1 gene.

We saw a 17-month-old boy with moderate psychomotor retardation, and enzymatically diagnosed succinic semialdehyde dehydrogenase (SSADH) deficiency. After extracting mRNA and genomic DNA from his cultured lymphoblasts, we analyzed the entire coding region of the ALDH5A1 gene using reverse transcription-polymerase chain reaction (RT-PCR) and genomic PCR followed by sequencing. He was demonstrated to be a compound heterozygote with two novel mutations (103-121 del and 1460T>A). The former leads to a frameshift and premature termination, and the latter is a missense mutation, V487E. Both mutations were also detected in the genomic DNA. Taken together with previous mutation reports, genetic heterogeneity was suspected for SSADH deficiency, and may account for the wide range of its phenotype.