Molecular Profiles of Breast Cancer in a Single Institution.

BACKGROUND/AIM: Historically, breast cancer has been treated according to an evaluation of biomarkers, such as the estrogen receptor and HER2 status. Recently, molecular profiling has been used to detect driver mutations and select anti-cancer treatment strategies. In addition to detecting pathogenic mutations, the total mutation count (tumor mutation burden) has been considered as another biomarker. MATERIALS AND METHODS: We performed molecular profiling of 143 breast cancer tissues obtained from resected tissues via surgical operation. RESULTS: Suspected germline mutations were detected in 10% of the patients with a higher somatic mutation ratio. CONCLUSION: As hypermutated breast cancers are more likely to benefit from certain anti-cancer treatment strategies, molecular profiling can be used as a biomarker.

Complex chromosomal rearrangements of human chromosome 21 in a patient manifesting clinical features partially overlapped with that of Down syndrome.

The chromosomal region critical in Down syndrome has long been analyzed through genotype-phenotype correlation studies using data from many patients with partial trisomy 21. Owing to that, a relatively small region of human chromosome 21 (35.9 ~ 38.0 Mb) has been considered as Down syndrome critical region (DSCR). In this study, microarray-based comparative genomic hybridization analysis identified complex rearrangements of chromosome 21 in a patient manifesting clinical features partially overlapped with that of Down syndrome. Although the patient did not show up-slanting palpebral fissures and single transverse palmar creases, other symptoms were consistent with Down syndrome. Rearrangements were analyzed by whole-genome sequencing using Nanopore long-read sequencing. The analysis revealed that chromosome 21 was fragmented into seven segments and reassembled by six connected points. Among 12 breakpoints, 5 are located within the short region and overlapped with repeated segments. The rearrangement resulted in a maximum gain of five copies, but no region showed loss of genomic copy numbers. Breakpoint-junctions showed no homologous region. Based on these findings, chromoanasynthesis was considered as the mechanism. Although the distal 21q22.13 region was not included in the aberrant regions, some of the genes located on the duplicated regions, SOD1, SON, ITSN1, RCAN1, and RUNX1, were considered as possible candidate genes for clinical features of the patient. We discussed the critical region for Down syndrome, with the literature review.

Analyses of breakpoint junctions of complex genomic rearrangements comprising multiple consecutive microdeletions by nanopore sequencing.

The widespread use of genomic copy number analysis has revealed many previously unknown genomic structural variations, including some which are more complex. In this study, three consecutive microdeletions were identified in the same chromosome by microarray-based comparative genomic hybridization (aCGH) analysis for a patient with a neurodevelopmental disorder. Subsequent fluorescence in situ hybridization (FISH) analyses unexpectedly suggested complicated translocations and inversions. For better understanding of the mechanism, breakpoint junctions were analyzed by nanopore sequencing, as a new long-read whole-genome sequencing (WGS) tool. The results revealed a new chromosomal disruption, giving rise to four junctions in chromosome 7. According the sequencing results of breakpoint junctions, all junctions were considered as the consequence of multiple double-strand breaks and the reassembly of DNA fragments by nonhomologous end-joining, indicating chromothripsis. KMT2E, located within the deletion region, was considered as the gene responsible for the clinical features of the patient. Combinatory usage of aCGH and FISH analyses would be recommended for interpretation of structural variations analyzed through WGS.

Mutations in NSD1 and NFIX in Three Patients with Clinical Features of Sotos Syndrome and Malan Syndrome.

Mutations in nuclear receptor SET domain-containing protein 1 gene ( NSD1 ) are related to Sotos syndrome, which is characterized by overgrowth, macrocephaly, distinctive features, and neurodevelopmental disabilities. On the other hand, mutations in the nuclear factor I/X gene ( NFIX ) can lead to Malan syndrome, also known as Sotos-like syndrome, or to the Marshall-Smith syndrome. In this study, using next generation sequencing (NGS), we identified de novo mutations in NSD1 and NFIX in three patients with developmental disabilities associated with overgrowth or macrocephaly. Overall, we confirmed that clinical entities of congenital malformation syndromes can be expanded by molecular diagnoses via NGS.

Familial 9q33q34 microduplication in siblings with developmental disorders and macrocephaly.

Because several genes responsible for epileptic encephalopathy are located on the 9q33q34 region, patients with chromosomal deletions of this region often show intractable epilepsy and neurodevelopmental disability. Contrary to these findings, chromosomal duplications of this region have never been reported previously. We identified a first case of 9q33q34 microduplications in siblings associated with developmental disorders and macrocephaly. Their mother was a mosaic carrier of this duplication. Duplicated regions involved STXBP1; the gene related to epileptic encephalopathy. Neurological features including developmental delay and macrocephaly observed in the present siblings may be derived from the extra-copy of STXBP1.

A novel CASK mutation identified in siblings exhibiting developmental disorders with/without microcephaly.

The calcium/calmodulin-dependent serine protein kinase gene (CASK) mutations are associated with various neurological disorders; a syndrome of intellectual disability (ID) and microcephaly with pontine and cerebellar hypoplasia (MICPCH), FG syndrome, X-linked ID with/without nystagmus, epileptic encephalopathy, and autistic spectrum disorder (ASD). Next generation sequencing was performed to elucidate genetic causes in siblings exhibiting developmental disorders, and a novel CASK mutation, c.1424G>T (p.Ser475Ile), was detected in a male patient with ID, ASD, and microcephaly. Radiological examination of his brain showed no structural abnormality. The identified mutation was shared with the healthy mother and a younger sister exhibiting ASD. Although the mother showed a skewed X-chromosome inactivation (XCI) pattern, the sister showed a paradoxical XCI pattern. This would explain why this sister possessed a normal intellectual level, but showed the same ASD symptoms as the affected brother. A novel CASK mutation was identified in two siblings with ID and/or ASD, suggesting a relationship between the CASK mutation and ASD. Recently performed large molecular cohorts for patients with developmental disorders suggest that CASK is one of the genes related to developmental disorders. For better understanding of genotype-phenotype correlation in ASD cases with CASK mutations, more information should be accumulated.

A novel TUBB4A mutation G96R identified in a patient with hypomyelinating leukodystrophy onset beyond adolescence.

The tubulin beta-4A gene (TUBB4A) is associated with two different clinical conditions, dystonia type 4 (DYT4) and hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). We identified a novel TUBB4A mutation, c.286G>A (p.G96R), in an adult male patient who suffered neurological symptoms beyond adolescence. This patient shows intermediate clinical features between DYT4 and H-ABC, suggesting that the TUBB4A disorder would constitute a spectrum disorder.

A 15q14 microdeletion involving MEIS2 identified in a patient with autism spectrum disorder.

We describe a 9-year-old male patient with a 15q14 microdeletion including MEIS2. The patient was born with a ventricular septal defect and submucosal cleft. Mild developmental disability and autism spectrum disorder diagnosed in childhood were also considered to be consequences of MEIS2 haploinsufficiency. The relatively mild developmental delay and lack of additional phenotypic features in this patient indicate that only MEIS2 plays an important role in the observed phenotypic features in the heterozygous state.

MED13L haploinsufficiency syndrome: A de novo frameshift and recurrent intragenic deletions due to parental mosaicism.

MED13L haploinsufficiency syndrome is a clinical condition manifesting intellectual disability and developmental delay in association with various complications including congenital heart defects and dysmorphic features. Most of the previously reported patients showed de novo loss-of-function mutations in MED13L. Additional three patients with MED13L haploinsufficiency syndrome were identified here in association with rare complications. One patient had a de novo deletion (c.257delT) and T2-weighted high intensity in the occipital white matter on magnetic resonance imaging. Two siblings exhibited an intragenic deletion involving exons 3-14, which led to an in-frame deletion in MED13L. The deletion was inherited from their carrier mother who possessed low frequency mosaicism. The older sister of the siblings showed craniosynostosis; this condition has never been reported in patients with MED13L haploinsufficiency syndrome. Dysmorphic features were observed in these patients; however, most of the findings were nonspecific. Further information would be necessary to understand this clinical condition better.

Concurrent occurrence of an inherited 16p13.11 microduplication and a de novo 19p13.3 microdeletion involving MAP2K2 in a patient with developmental delay, distinctive facial features, and lambdoid synostosis.

A female patient presented with developmental delay, distinctive facial features, and congenital anomalies, including a heart defect and premature lambdoid synostosis. The patient showed a paternally inherited 16p13.11 microduplication and a de novo 19p13.3 microdeletion involving the mitogen-activated protein kinase kinase 2 gene (MAP2K2), in which mutations cause the cardio-facio-cutaneous (CFC) syndrome. Reports of patients with overlapping 19p13.3 microdeletions of this region describe similar clinical manifestations including distinctive facial features: prominent forehead, horizontal/down-slanting palpebral fissures, long midface, pointed chin/angular jaw, sparse eyebrows, and underdeveloped cheekbones. Some of these findings overlapped to that of the patients with 16p13.11 microduplications and CFC syndrome. Although craniosynostosis was occasionally observed in patients with dominant-negative mutations in RAS/MAP kinase signaling genes (RASopathies) related to CFC syndrome, it was also reported in two patients with 16p13.11 microduplications. Genetic contributions of both chromosomal aberrations were discussed.

Loss-of-function mutations and global rearrangements in GPC3 in patients with Simpson-Golabi-Behmel syndrome.

Simpson-Golabi-Behmel syndrome is a congenital malformation syndrome associated with mutations in GPC3, which is located in the Xq26 region. Three new loss-of-function mutations and a global X-chromosome rearrangement involving GPC3 were identified. A female sibling of the patient, who presented with a cleft palate and hepatoblastoma, carries the same chromosomal rearrangement and a paradoxical pattern of X-chromosome inactivation. These findings support variable GPC3 alterations, with a possible mechanism in female patients.

Challenges in detecting genomic copy number aberrations using next-generation sequencing data and the eXome Hidden Markov Model: a clinical exome-first diagnostic approach.

Next-generation sequencing (NGS) is widely used for the detection of disease-causing nucleotide variants. The challenges associated with detecting copy number variants (CNVs) using NGS analysis have been reported previously. Disease-related exome panels such as Illumina TruSight One are more cost-effective than whole-exome sequencing (WES) because of their selective target regions (~21% of the WES). In this study, CNVs were analyzed using data extracted through a disease-related exome panel analysis and the eXome Hidden Markov Model (XHMM). Samples from 61 patients with undiagnosed developmental delays and 52 healthy parents were included in this study. In the preliminary study to validate the constructed XHMM system (microarray-first approach), 34 patients who had previously been analyzed by chromosomal microarray testing were used. Among the five CNVs larger than 200 kb that were considered as non-pathogenic CNVs and were used as positive controls, four CNVs was successfully detected. The system was subsequently used to analyze different samples from 27 patients (NGS-first approach); 2 of these patients were successfully diagnosed as having pathogenic CNVs (an unbalanced translocation der(5)t(5;14) and a 16p11.2 duplication). These diagnoses were re-confirmed by chromosomal microarray testing and/or fluorescence in situ hybridization. The NGS-first approach generated no false-negative or false-positive results for pathogenic CNVs, indicating its high sensitivity and specificity in detecting pathogenic CNVs. The results of this study show the possible clinical utility of pathogenic CNV screening using disease-related exome panel analysis and XHMM.

A de novo microdeletion in a patient with inner ear abnormalities suggests that the 10q26.13 region contains the responsible gene.

Microdeletions in the 10q26.1 region are related to intellectual disability, growth delay, microcephaly, distinctive craniofacial features, cardiac defects, genital abnormalities and inner ear abnormalities. The genes responsible for inner ear abnormalities have been narrowed to fibroblast growth factor receptor 2 gene (FGFR2), H6 family homeobox 2 gene (HMX2) and H6 family homeobox 3 gene (HMX3). An additional patient with distinctive craniofacial features, congenital deafness and balance dysfunctions showed a de novo microdeletion of 10q26.11q26.13, indicating the existence of a gene responsible for inner ear abnormalities in this region.

Novel variants of the SHANK3 gene in Japanese autistic patients with severe delayed speech development.

The 22q13.3 deletion syndrome is characterized by a significant delay in language development, mental retardation, hypotonia, and autistic features. Cumulative evidence has shown that haploinsufficiency of the SHANK3 gene is a major cause of the neurological symptoms of the 22q13.3 deletion syndrome. Shank3, a multidomain protein containing the SH3 and PDZ domains, is thought to play an important role in the formation and function of synapses in the developing brain. In this study, we analyzed the SHANK3 gene in 128 autistic patients with manifestations similar to those seen in the 22q13.3 deletion syndrome. The results showed a 6-amino acid deletion upstream of the SH3 domain, a missense variant (arginine to histidine at amino acid position 656) in the PDZ domain, and the insertion or deletion of a repeated 10-bp GC sequence located 9-bp downstream from the 3' end of exon 11. None of these variants was found in 228 controls.

Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor.

A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons.

Slo2 sodium-activated K+ channels bind to the PDZ domain of PSD-95.

Slo2 channels are a type of sodium-activated K+ channels and possess a typical PDZ binding motif at the carboxy-terminal end. Thus, we investigated whether Slo2 channels bind to PSD-95, because it is well known that other types of K+ channels, voltage-gated and inward rectifier K+ channels, bind to PSD-95 via the PDZ binding motif and are involved in excitatory synaptic transmission. By using an extract prepared from cultured neocortical neurons, we demonstrated a biochemical interaction between mSlo2 channels and PSD-95, and a mutational analysis revealed that mSlo2 channels bound to the first PDZ domain of PSD-95 via the PDZ binding motif. To investigate the expression of mSlo2 protein in primary neocortical neurons, we raised anti-mSlo2 channel antibody and immunostained neocortical neurons. The immunocytochemical study showed that mSlo2 channels partly colocalized with PSD-95 in mouse neocortical neurons.