An evaluation of the challenges to developing tumor BRCA1 and BRCA2 testing methodologies for clinical practice.

Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Tumor BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin-fixed paraffin embedded (FFPE), the tumor genome is complex, and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumors to compare different approaches to identify clinically important variants within FFPE tumor DNA samples. This was not a proficiency study but an inter-laboratory comparison to identify common issues. Each laboratory received the same tumor DNA samples ranging in genotype, quantity, quality, and variant allele frequency (VAF). Each laboratory performed their preferred next-generation sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification, or insufficient DNA. The use of hybridization capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardization for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies.

New insights into the performance of human whole-exome capture platforms.

Whole exome sequencing (WES) is increasingly used in research and diagnostics. WES users expect coverage of the entire coding region of known genes as well as sufficient read depth for the covered regions. It is, however, unknown which recent WES platform is most suitable to meet these expectations. We present insights into the performance of the most recent standard exome enrichment platforms from Agilent, NimbleGen and Illumina applied to six different DNA samples by two sequencing vendors per platform. Our results suggest that both Agilent and NimbleGen overall perform better than Illumina and that the high enrichment performance of Agilent is stable among samples and between vendors, whereas NimbleGen is only able to achieve vendor- and sample-specific best exome coverage. Moreover, the recent Agilent platform overall captures more coding exons with sufficient read depth than NimbleGen and Illumina. Due to considerable gaps in effective exome coverage, however, the three platforms cannot capture all known coding exons alone or in combination, requiring improvement. Our data emphasize the importance of evaluation of updated platform versions and suggest that enrichment-free whole genome sequencing can overcome the limitations of WES in sufficiently covering coding exons, especially GC-rich regions, and in characterizing structural variants.

Hb Bakersfield (HBA1: c.151_152insGGAGCC): The Insertion of Arg-His Between Codons 49 and 50 of the α1-Globin Chain Leads to Increased Oxygen Affinity.

We describe an insertion variant on the α1-globin gene (HBA1) identified in a 49-year-old woman of Jurassian ancestry presenting with macrocytosis and erythrocytosis. The variant resulted in a peak of 15.5% of the total hemoglobin (Hb) on high performance liquid chromatography (HPLC). Stability and oxygen affinity testing revealed that the variant was stable and had an increased oxygen affinity. Molecular genetic testing detected the heterozygous sequence variant Hb Bakersfield [α50(CE8)His→0; Arg-Ser-His- inserted between 49(CE7) and 51(CE9) of α1; HBA1: c.151_152insGGAGCC (p.Ser50_His51insArgSer)] in the index patient, one of her sons, as well as in two of her grandchildren, who showed a similar hematological pattern.

Detection of germline rearrangements in patients with α- and ß-thalassemia using high resolution array CGH.

Approximately 80% of α-thalassemia mutations are deletions in the α-globin cluster on chromosome 16 and about 10% of ß-thalassemia mutations are deletions in the ß-globin gene cluster on chromosome 11. Larger deletions involving the ß-globin gene cluster lead to (δß)-, (γδß)-, (εγδß)-thalassemia, or hereditary persistence of fetal hemoglobin (HPFH). Array comparative genomic hybridization (CGH) was applied to screen for deletions in the α- and ß-globin gene clusters not detected by routine gap-PCR. In total, in 13 patients with hypochromia and inclusion bodies (IBs) the α-globin gene cluster was analyzed and in 13 patients with increased fetal hemoglobin levels with or without hypochromia the ß-globin gene cluster was examined. All samples were subsequently investigated by multiplex ligation-dependent probe amplification (MLPA). In 9 out of 13 patients deletions of the α-globin gene cluster were identified; 5 of these deletions remove the entire α-globin cluster and extend to the telomere. Additional sequencing of the remaining 4 patients revealed polyadenylation mutation in 1 of them. 7 deletions were identified in the ß-globin gene cluster in 13 patients. Additional sequencing of the remaining 6 patients revealed mutations in one of the γ-globin gene promoters in 3 of them and a KLF1-mutation in 1 of them. Array CGH is a reliable method to screen for deletions in thalassemia and hemoglobinopathy. The method offers the advantage of a high resolution with the possibility to characterize breakpoints on sequence level.

A New de novo BRCA1 Mutation in a Young Breast Cancer Patient: A Case Report.

Background: BRCA1 and BRCA2 genes represent the most investigated breast and ovarian cancer predisposition genes. Ten cases of pathogenic de novo BRCA1 variations and six cases of pathogenic de novo BRCA2 variation have been reported at present. Here, we report a new case of a de novo BRCA1 gene mutation. Case Presentation: A 30-year-old woman with no health issues and no family history for hereditary breast and ovarian cancer was diagnosed with a hormone receptor positive/HER2 negative invasive breast cancer. Genetic testing revealed a pathogenic variant in BRCA1 (c.4065_4068delTCAA) which was not found in her parents or sister. Conclusion: We report a new case of de novo BRCA1 mutation, confirmed by repeated germline testing of the index patient and her parents. The published BRCA1/2 de novo mutation rate is low. This is probably due - in part - to the strict testing criteria.

Microdeletion 19p13.2 in an almost 5-year-old boy.

Deletions of the short arm of chromosome 19 are rarely found by conventional cytogenetic techniques. This region has a high gene density and this is likely the reason why deletions in this region are associated with a severe phenotype. Since the implementation of modern high-resolution SNP- and CGH-array techniques more cases have been reported. Here, we present an almost 5-year-old boy with intellectual disability, minor dysmorphisms, febrile seizures, and a de novo deletion of 834.2 kb on 19p13.2 encompassing 32 genes. The deletion was found by the Illumina Infinium HD Human1M-Duo v1 BeadChip SNP-array and confirmed by the NimbleGen Human CGH 2.1M Whole Genome Tiling v2.0D oligonucleotide array. PCR amplification of the junction fragment and subsequent sequencing defined the breakpoints and indicated that formation was mediated by non-allelic homologous recombination (NAHR). The phenotype of our patient shows that microrearrangements even at gene-dense chromosomes may result in mild clinical consequences.

Reduced expression by SETBP1 haploinsufficiency causes developmental and expressive language delay indicating a phenotype distinct from Schinzel-Giedion syndrome.

BACKGROUND: Mutations of the SET binding protein 1 gene (SETBP1) on 18q12.3 have recently been reported to cause Schinzel-Giedion syndrome (SGS). As rare 18q interstitial deletions affecting multiple genes including SETBP1 correlate with a milder phenotype, including minor physical anomalies and developmental and expressive speech delay, mutations in SETBP1 are thought to result in a gain-of-function or a dominant-negative effect. However, the consequence of the SETBP1 loss-of-function has not yet been well described. METHODS: Microarray-based comparative genomic hybridisation (aCGH) analyses were performed to identify genetic causes for developmental and expressive speech delay in two patients. SETBP1 expression in fibroblasts obtained from one of the patients was analysed by real-time RT-PCR and western blotting. A cohort study to identify nucleotide changes in SETBP1 was performed in 142 Japanese patients with developmental delay. RESULTS: aCGH analyses identified submicroscopic deletions of less than 1 Mb exclusively containing SETBP1. Both patients show global developmental, expressive language delay and minor facial anomalies. Decreased expression of SETBP1 was identified in the patient's skin fibroblasts. No pathogenic mutation of SETBP1 was identified in the cohort study. CONCLUSION: SETBP1 expression was reduced in a patient with SETBP1 haploinsufficiency, indicating that the SETBP1 deletion phenotype is allele dose sensitive. In correlation with the exclusive deletion of SETBP1, this study delimits a milder phenotype distinct from SGS overlapping with the previously described phenotype of del(18)(q12.2q21.1) syndrome including global developmental, expressive language delay and distinctive facial features. These findings support the hypothesis that mutations in SETBP1 causing SGS may have a gain-of-function or a dominant-negative effect, whereas haploinsufficiency or loss-of-function mutations in SETBP1 cause a milder phenotype.

Update Swiss guideline for counselling and testing for predisposition to breast, ovarian, pancreatic and prostate cancer.

This paper presents the Swiss guideline for genetic counselling and testing of individuals with an increased probability for carrying mutations in high risk cancer predisposition genes, particularly BRCA1 and BRCA2. It aims to help providers of genetic counselling to identify valuable candidates for testing and serves as a basis for reimbursement claims to Swiss insurance companies.

Homozygous nonsense mutation in WNT10B and sporadic split-hand/foot malformation (SHFM) with autosomal recessive inheritance.

Split-hand/foot malformation (SHFM) is a limb malformation affecting the central rays of the hands and/or feet. Isolated SHFM occurs within families but more often sporadically. Since most families with more than one patient show dominant inheritance with reduced penetrance, sporadic SHFM is generally considered to be due to dominantly inherited new mutations. Recently, recessive inheritance of SHFM was proposed in a highly consanguineous family with a homozygous missense mutation in WNT10B. Nevertheless, the assumption of a second locus was necessary to explain the observed phenotypes in this family. To date, no other family and no case of sporadic SHFM with WNT10B mutations are known. By examining WNT10B in a patient with sporadic SHFM, we identified a homozygous 4-bp duplication resulting in a premature termination codon. Nine heterozygous relatives show no sign of SHFM. These findings have profound implications for genetic counseling. Obviously, sporadic SHFM may show recessive rather than dominant inheritance resulting in a 25% recurrence risk for sibs instead of a very low-recurrence risk as generally presumed. Likewise, there is a very low-recurrence risk for offspring of patients (unless there is consanguinity) instead of an estimated risk between 30% and 50%. It can be concluded that sporadic SHFM is not always a dominant trait. To determine the recurrence risk, patients affected with sporadic SHFM should be tested for mutations in WNT10B.

Deletion of 7q11.21-q11.23 and infantile spasms without deletion of MAGI2.

We report on the clinical and cytogenetic findings and on the array-based characterization of an interstitial 7q11.21-q11.23 deletion initially recognized by standard karyotyping in a 15-month-old female patient. Beginning at the age of 3 months and 2 weeks the patient had severe infantile spasms. Recently, it was reported that infantile spasms are associated with deletion of the MAGI2 gene on chromosome 7q11.23. Nevertheless, not all patients reported with deletions of MAGI2 developed infantile spasms and at least one reported patient with a deletion 7q11.23 without missing the MAGI2 gene was diagnosed with infantile spasms. Molecular karyotyping of our patient confirmed a large 13 Mb deletion encompassing the 7q11.21-q11.23 region without involvement of MAGI2. Critical review of published data and the results of our patient underline the importance to map precisely the deletion boundaries of further patients to reevaluate the significance of MAGI2 hemizygosity in the pathogenesis of infantile spasms.

Interstitial deletion 1q42 in a patient with agenesis of corpus callosum: Phenotype-genotype comparison to the 1q41q42 microdeletion suggests a contiguous 1q4 syndrome.

Interstitial deletions of 1q4 are rare and present with different deletion breakpoints and variable phenotype. We report on the clinical and molecular cytogenetic findings in a girl with minor anomalies, midline defects including prenatally ascertained agenesis of the corpus callosum, epilepsy and developmental delay. A de novo 5.45 Mb deletion almost exclusively located within 1q42 was found to cause this phenotype, which shows significant overlap with the microdeletion 1q41q42 syndrome reported in a few patients except for the agenesis of the corpus callosum. However, deletions in patients with the 1q41q42 syndrome mainly extend into the 1q41 region with a region of overlap including the DISP1 gene involved in the SHH pathway, which is not part of the 1q42 deletion in our patient. We suggest that an interaction of genes involved in pathways of embryonic development rather than haploinsufficiency of single genes in the so-called critical regions is causing complex malformation syndromes due to cytogenetic microaberrations in the 1q4 region.

Familial 14.5 Mb interstitial deletion 13q21.1-13q21.33: clinical and array-CGH study of a benign phenotype in a three-generation family.

We report on the clinical and cytogenetic findings as well as the array-based characterization of an interstitial familial 13q21 deletion initially recognized by standard karyotyping. Although 13q deletions are known to imply a wide variability of clinical consequences, the deletion carriers of the familial deletion in three generations did not reveal a relevant phenotype. The breakpoints and the deletion size in all three carrier individuals were determined by molecular karyotyping confirming a large 14.5 Mb deletion encompassing the 13q21.1-13q21.33 region identical in all three carriers. Gene paucity and the lack of dosage-sensitive genes in the delineated region might explain the apparently innocuous nature of this chromosomal anomaly. The example of this family presents evidence for describing the chromosomal region 13q21.1-13q21.33 as a large euchromatic variant or benign copy number variation without phenotypic consequences. Our data underline the importance of a phenogenetic approach combining clinical and laboratory evidence in the interpretation of segmental chromosomal anomalies especially in genetic counseling related to prenatal diagnosis.

[Impact of tumorcytogenetics in tumor diagnostics]. / Bedeutung der Tumorzytogenetik in der Tumordiagnostik.

Most tumors are caused by acquired somatic mutations. For half a century now chromosomal anomalies in hematological neoplasia and to a much less extent in solid tumors are investigated by conventional cytogenetics. During all this time the significance of genetic factors for tumor classification increased continuously. Especially for haematological neoplasia, besides <> cytogenetic parameters, molecular genetic factors are becoming more and more important. For technical and analytical reasons conventional cytogenetics does not play a role in routine diagnostics of solid tumors. However, well known chromosome aberrations are increasingly investigated by fluorescence-in-situ Hybridization (FISH) and by molecular genetic methods. In the future microarray-based methods will become more and more important in the diagnostics of various tumors.

Recurrence risk in de novo structural chromosomal rearrangements.

According to the textbook of Gardner and Sutherland [2004], the standard on genetic counseling for chromosome abnormalities, the recurrence risk of de novo structural or combined structural and numeric chromosome rearrangements is less than 0.5-2% and takes into account recurrence by chance, gonadal mosaicism, and somatic-gonadal mosaicism. However, these figures are roughly estimated and neither any systematic study nor exact or evidence-based risk calculations are available. To address this question, an extensive literature search was performed and surprisingly only 29 case reports of recurrence of de novo structural or combined structural and numeric chromosomal rearrangements were found. Thirteen of them were with a trisomy 21 due to an i(21q) replacing one normal chromosome 21. In eight of them low-level mosaicism in one of the parents was found either in fibroblasts or in blood or in both. As a consequence of the low number of cases and theoretical considerations (clinical consequences, mechanisms of formation, etc.), the recurrence risk should be reduced to less than 1% for a de novo i(21q) and to even less than 0.3% for all other de novo structural or combined structural and numeric chromosomal rearrangements. As the latter is lower than the commonly accepted risk of approximately 0.3% for indicating an invasive prenatal diagnosis and as the risk of abortion of a healthy fetus after chorionic villous sampling or amniocentesis is higher than approximately 0.5%, invasive prenatal investigation in most cases is not indicated and should only be performed if explicitly asked by the parents subsequent to appropriate genetic counseling.

TRIB1 overexpression in acute myeloid leukemia.

In this report, an AML patient with an additional ring chromosome 8 was investigated in detail. Fluorescence in situ hybridization showed high amplification of MYC on the additional ring chromosome and signals on both chromosome 8 homologues. In addition to the amplified segment from 8q24, no additional chromosomal aberration was found by array comparative genomic hybridization. Real-time reverse-transcription polymerase chain reaction analysis revealed that MYC and TRIB1 (a gene also localized on 8q24, close to MYC) were clearly overexpressed when compared with healthy donors, acute myeloid leukemia patients without MYC amplification, chronic myeloid leukemia patients, and acute lymphatic leukemia patients. These results may indicate a key role of TRIB1 (and MYC) overexpression in the pathogenesis of a certain AML subtype.

The Application of Next-Generation Sequencing for Mutation Detection in Autosomal-Dominant Hereditary Hearing Impairment.

OBJECTIVE: Identification of the causative mutation using next-generation sequencing in autosomal-dominant hereditary hearing impairment, as mutation analysis in hereditary hearing impairment by classic genetic methods, is hindered by the high heterogeneity of the disease. PATIENTS: Two Swiss families with autosomal-dominant hereditary hearing impairment. INTERVENTION: Amplified DNA libraries for next-generation sequencing were constructed from extracted genomic DNA, derived from peripheral blood, and enriched by a custom-made sequence capture library. Validated, pooled libraries were sequenced on an Illumina MiSeq instrument, 300 cycles and paired-end sequencing. Technical data analysis was performed with SeqMonk, variant analysis with GeneTalk or VariantStudio. The detection of mutations in genes related to hearing loss by next-generation sequencing was subsequently confirmed using specific polymerase-chain-reaction and Sanger sequencing. MAIN OUTCOME MEASURE: Mutation detection in hearing-loss-related genes. RESULTS: The first family harbored the mutation c.5383+5delGTGA in the TECTA-gene. In the second family, a novel mutation c.2614-2625delCATGGCGCCGTG in the WFS1-gene and a second mutation TCOF1-c.1028G>A were identified. CONCLUSION: Next-generation sequencing successfully identified the causative mutation in families with autosomal-dominant hereditary hearing impairment. The results helped to clarify the pathogenic role of a known mutation and led to the detection of a novel one. NGS represents a feasible approach with great potential future in the diagnostics of hereditary hearing impairment, even in smaller labs.

Guidance Statement On BRCA1/2 Tumor Testing in Ovarian Cancer Patients.

The approval, in 2015, of the first poly (adenosine diphosphate-ribose) polymerase inhibitor (PARPi; olaparib, Lynparza) for platinum-sensitive relapsed high-grade ovarian cancer with either germline or somatic BRCA1/2 deleterious mutations is changing the way that BRCA1/2 testing services are offered to patients with ovarian cancer. Ovarian cancer patients are now being referred for BRCA1/2 genetic testing for treatment decisions, in addition to familial risk estimation, and irrespective of a family history of breast or ovarian cancer. Furthermore, testing of tumor samples to identify the estimated 3%-9% of patients with somatic BRCA1/2 mutations who, in addition to germline carriers, could benefit from PARPi therapy is also now being considered. This new testing paradigm poses some challenges, in particular the technical and analytical difficulties of analyzing chemically challenged DNA derived from formalin-fixed, paraffin-embedded specimens. The current manuscript reviews some of these challenges and technical recommendations to consider when undertaking BRCA1/2 testing in tumor tissue samples to detect both germline and somatic BRCA1/2 mutations. Also provided are considerations for incorporating genetic analysis of ovarian tumor samples into the patient pathway and ethical requirements.

Lymphadenopathy driven by TCR-Vγ8Vδ1 T-cell expansion in FAS-related autoimmune lymphoproliferative syndrome.

FAS-dependent apoptosis in Vδ1 T cells makes the latter possible culprits for the lymphadenopathy observed in patients with FAS mutations.Rapamycin and methylprednisolone resistance should prompt clinicians to look for Vδ1 T cell proliferation in ALPS-FAS patients.

Autism and intellectual disability in a patient with two microdeletions in 6q16: a contiguous gene deletion syndrome?

BACKGROUND: Copy number variations play a significant role in the aetiology of developmental disabilities including non-syndromic intellectual disability and autism. CASE PRESENTATION: We describe a 19-year old patient with intellectual disability and autism for whom chromosomal microarray (CMA) analysis showed the unusual finding of two de novo microdeletions in cis position on chromosome 6q16.1q16.2 and 6q16.3. The two deletions span 10 genes, including FBXL4, POU3F2, PRDM13, CCNC, COQ3 and GRIK2. We compared phenotypes of patients with similar deletions and looked at the involvement of the genes in neuronal networks in order to determine the pathogenicity of our patient's deletions. CONCLUSIONS: We suggest that both deletions on 6q are causing his disease phenotype since they harbour several genes which are implicated in pathways of neuronal development and function. Further studies regarding the interaction between PRDM13 and GRIK2 specifically may be interesting.