Can the Synergic Contribution of Multigenic Variants Explain the Clinical and Cellular Phenotypes of a Neurodevelopmental Disorder?

We describe an infant female with a syndromic neurodevelopmental clinical phenotype and increased chromosome instability as cellular phenotype. Genotype characterization revealed heterozygous variants in genes directly or indirectly linked to DNA repair: a de novo X-linked HDAC8 pathogenic variant, a paternally inherited FANCG pathogenic variant and a maternally inherited BRCA2 variant of uncertain significance. The full spectrum of the phenotype cannot be explained by any of the heterozygous variants on their own; thus, a synergic contribution is proposed. Complementation studies showed that the FANCG gene from the Fanconi Anaemia/BRCA (FA/BRCA) DNA repair pathway was impaired, indicating that the variant in FANCG contributes to the cellular phenotype. The patient’s chromosome instability represents the first report where heterozygous variant(s) in the FA/BRCA pathway are implicated in the cellular phenotype. We propose that a multigenic contribution of heterozygous variants in HDAC8 and the FA/BRCA pathway might have a role in the phenotype of this neurodevelopmental disorder. The importance of these findings may have repercussion in the clinical management of other cases with a similar synergic contribution of heterozygous variants, allowing the establishment of new genotype–phenotype correlations and motivating the biochemical study of the underlying mechanisms.

Genomic Features of Chinese Small Cell Lung Cancer

Background: Small cell lung cancer (SCLC) is an aggressive disease with poor survival. Although molecular and clinical characteristics have been established for SCLC in western patients, limited investigation has been performed for Chinese SCLC patients.Objective: In this study, we investigated the genomic features of Chinese SCLC patients.Methods: A total of 75 SCLC patients were enrolled. Genomic alterations in 618 selected genes were analyzed by targeted next-generation sequencing.Results: Here, we showed that TP53 (77.30%) and RB1 (30.70%) were the most prevalent genes, followed by KMT2D, ALK, LRP1B, EGFR, NOTCH3, AR, CREBBP, ROS1, and BRCA2. And the most common genetic alterations were enriched in the cell cycle signaling pathway (84.00%) of Chinese SCLC patients. DNA damage repair (DDR) pathway analysis showed that the most frequently enriched DDR pathways were fanconi anaemia (FA, 29.41%) and homology recombination (HR, 21.57%). Notably, 9.33% SCLC patients in our cohort had pathogenic or likely pathogenic germline gene variants. Compared with the U Cologne cohort, a higher prevalence in EGFR, AR, BRCA2, TSC1, ATXN3, MET, MSH2, ERBB3 and FOXA1 were found in our cohort; while compared to the data from the Johns Hopkins cohort, a higher mutated frequency in TP53, KMT2D, ALK, and EGFR were found in our cohort. Moreover, a significant association was found between high tumor mutation burden (TMB) and mutations involved in TP53, CREBBP, EPHA3, KMT2D, ALK and RB1. Approximately 33.33% of patients with SCLC harbored at least one actionable alteration annotated by OncoKB, of which one patient had alterations of level 1; seventeen patients had level 3; fifteen patients possessed level 4.Conclusion: Our data might provide an insightful meaning in targeted therapy for Chinese SCLC patients.

Inference of tissue relative proportions of the breast epithelial cell types luminal progenitor, basal, and luminal mature

Single-cell analysis has revolutionised genomic science in recent years. However, due to cost and other practical considerations, single-cell analyses are impossible for studies based on medium or large patient cohorts. For example, a single-cell analysis usually costs thousands of euros for one tissue sample from one volunteer, meaning that typical studies using single-cell analyses are based on very few individuals. While single-cell genomic data can be used to examine the phenotype of individual cells, cell-type deconvolution methods are required to track the quantities of these cells in bulk-tissue genomic data. Hormone receptor negative breast cancers are highly aggressive, and are thought to originate from a subtype of epithelial cells called the luminal progenitor. In this paper, we show how to quantify the number of luminal progenitor cells as well as other epithelial subtypes in breast tissue samples using DNA and RNA based measurements. We find elevated levels of cells which resemble these hormone receptor negative luminal progenitor cells in breast tumour biopsies of hormone receptor negative cancers, as well as in healthy breast tissue samples from BRCA1 (FANCS) mutation carriers. We also find that breast tumours from carriers of heterozygous mutations in non-BRCA Fanconi Anaemia pathway genes are much more likely to be hormone receptor negative. These findings have implications for understanding hormone receptor negative breast cancers, and for breast cancer screening in carriers of heterozygous mutations of Fanconi Anaemia pathway genes.

A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene

Background Familial ovarian cancer (OC) cases not harbouring pathogenic variants in either of the BRCA1 and BRCA2 OC-predisposing genes, which function in homologous recombination (HR) of DNA, could involve pathogenic variants in other DNA repair pathway genes. Methods Whole exome sequencing was used to identify rare variants in HR genes in a BRCA1 and BRCA2 pathogenic variant negative OC family of French Canadian (FC) ancestry, a population exhibiting genetic drift. OC cases and cancer-free individuals from FC and non-FC populations were investigated for carrier frequency of FANCI c.1813C>T; p.L605F, the top-ranking candidate. Gene and protein expression were investigated in cancer cell lines and tissue microarrays, respectively. Results In FC subjects, c.1813C>T was more common in familial (7.1%, 3/42) than sporadic (1.6%, 7/439) OC cases (P = 0.048). Carriers were detected in 2.5% (74/2950) of cancer-free females though female/male carriers were more likely to have a first-degree relative with OC (121/5249, 2.3%; Spearman correlation = 0.037; P = 0.011), suggesting a role in risk. Many of the cancer-free females had host factors known to reduce risk to OC which could influence cancer risk in this population. There was an increased carrier frequency of FANCI c.1813C>T in BRCA1 and BRCA2 pathogenic variant negative OC families, when including the discovery family, compared to cancer-free females (3/23, 13%; OR = 5.8; 95%CI = 1.7–19; P = 0.005). In non-FC subjects, 10 candidate FANCI variants were identified in 4.1% (21/516) of Australian OC cases negative for pathogenic variants in BRCA1 and BRCA2, including 10 carriers of FANCI c.1813C>T. Candidate variants were significantly more common in familial OC than in sporadic OC (P = 0.04). Localization of FANCD2, part of the FANCI-FANCD2 (ID2) binding complex in the Fanconi anaemia (FA) pathway, to sites of induced DNA damage was severely impeded in cells expressing the p.L605F isoform. This isoform was expressed at a reduced level, destabilized by DNA damaging agent treatment in both HeLa and OC cell lines, and exhibited sensitivity to cisplatin but not to a poly (ADP-ribose) polymerase inhibitor. By tissue microarray analyses, FANCI protein was consistently expressed in fallopian tube epithelial cells and only expressed at low-to-moderate levels in 88% (83/94) of OC samples. Conclusions This is the first study to describe candidate OC variants in FANCI, a member of the ID2 complex of the FA DNA repair pathway. Our data suggest that pathogenic FANCI variants may modify OC risk in cancer families.

Gene therapy and editing in the treatment of hereditary blood disorders: Medical and ethical aspects

Gene therapy and gene editing are revolutionising the treatment of genetic diseases, most notably haematological disorders. This paper evaluates the use of both techniques in hereditary blood disorders. Many studies have been conducted in this field, especially with gene therapy, with very promising results in diseases such as haemophilia, certain haemoglobinopathies and Fanconi anaemia. The application of these techniques in clinical practice and the foreseeable development of these approaches in the coming years suggest that it might be useful to evaluate the results achieved thus far. It is also essential to reflect on the possible bioethical concerns raised by the use of both techniques, especially in terms of safety issues for patients, potential side effects, treatment duration, accessibility and cost of treatment, and the heritability of genetic changes produced if germline cells are used.

Optimization of Robust Diagnostic Strategy for Patients with Fanconi Anaemia (FA)- an Indian Perspective

Objective: Fanconi anaemia is a genotypically heterogeneous disease. A fast and reliable genetic diagnosis method helps is important for the clinical care of these patients. The objective of this study was to establish a strategy for expeditious molecular diagnosis for the Indian FA patients. Methods: Exome sequencing was performed for 119 FA patients on Illumina HiSeq X system and the data was analysed by Sentieon (v201808.01) to identify germline variants. Single nucleotide variants (SNVs) with an allele frequency of <3% in various population databases including the Indian population database were analysed and annotated using an in-house pipeline. Copy number variants (CNVs) were detected using ExomeDepth (v1.1.10). Long amplicon next-generation sequencing was performed for FANCA and FANCG genes. Gene dosage analysis was performed for the deletions identified by WES in the FANCT/UBE2T gene. Sanger sequencing was performed for the selected variants identified by WES. Results: SNVs associated with FA were identified in heterozygous states in 93 (78%) patients. Of the remaining 20 patients, 8 had homozygous deletions, and twelve were compound heterozygous with deletions and SNVs. We detected deletions in 20 patients: 7 with heterozygous and 12 with homozygous deletions in FANCA, and one with a homozygous deletion in FANCT (UBE2T). MLPA confirmed the deletions in the FANCA gene. The homozygous deletion in the FANCT gene was confirmed by the lack of amplification by PCR using the primers binding to the deleted region. By combining the SNVs and deletions, disease-causing genotypes were identified in 113 of 119 (95%) patients. A large number of our patients (81.5 %) were homozygous (Figure) due to the high rate of consanguinity in the population. FANCA was found to be the most frequently mutated gene in (57.5%) while mutations in FANCG gene accounted for 14.2% of our patients. FANCC mutations were found at a very low frequency (1.8%) in our patients. Although mutations in FANCL are rare in all the populations, 24 (21.2%) patients with FANCL mutations were identified in our study. A previously reported synonymous splicing mutation FANCL c.1092G>A;p.K364= was found in homozygous state in 22 (19.5%) patients. Two other FANCL mutations identified includes a missense mutation c.827C>T; p.Pro281Leu in the homozygous state in one patient and a nonsense mutation c.997C>T; p.Gln333Ter in the compound heterozygous state with the common FANCL c.1092G>A;p.K364= mutation in another patient. Mutations in rarely mutated FA genes included 1 patient each in FANCT/UBE2T and FANCI and two each in FANCJ/BRIP1 and FANCF gene. Based on the comprehensive genotype analysis, we could design an algorithm for FA in the Indian population. ~20% of the FA patients who have FANCL c.1092G>A;p.K364= mutation can be diagnosed by Sanger sequencing. MLPA can detect FANCA deletions (16.5% of the overall mutations). The results from these two tests can be obtained in 48 hours. For those who are negative for the mutations by these two methods, LA-NGS can detect SNVs in the FANCA and FANCG genes, which constitute >55% of the FA mutations in the population. We have tested this algorithm for expedited molecular diagnosis in 27 FA patients and found that the disease genotypes could be established in 94% of the patients in less than two weeks. Conclusion/Clinical applicability: Exome sequencing identified several novel mutations in the FA pathway genes in the FA patients. A cost-effective and time-saving algorithm was explicitly established for molecular diagnosis of FA in the Indian population. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Fanconi Anaemia, Childhood Cancer and the BRCA Genes

Fanconi anaemia (FA) is an inherited chromosomal instability disorder characterised by congenital and developmental abnormalities and a strong cancer predisposition. In less than 5% of cases FA can be caused by bi-allelic pathogenic variants (PGVs) in BRCA2/FANCD1 and in very rare cases by bi-allelic PGVs in BRCA1/FANCS. The rarity of FA-like presentation due to PGVs in BRCA2 and even more due to PGVs in BRCA1 supports a fundamental role of the encoded proteins for normal development and prevention of malignant transformation. While FA caused by BRCA1/2 PGVs is strongly associated with distinct spectra of embryonal childhood cancers and AML with BRCA2-PGVs, and also early epithelial cancers with BRCA1 PGVs, germline variants in the BRCA1/2 genes have also been identified in non-FA childhood malignancies, and thereby implying the possibility of a role of BRCA PGVs also for non-syndromic cancer predisposition in children. We provide a concise review of aspects of the clinical and genetic features of BRCA1/2-associated FA with a focus on associated malignancies, and review novel aspects of the role of germline BRCA2 and BRCA1 PGVs occurring in non-FA childhood cancer and discuss aspects of clinical and biological implications.

72 Increasing Radiotherapy Induced DNA Damage by Inhibiting Repair Proteins

Introduction Fanconi anaemia (FA) is a rare genetic disorder displaying higher incidences of head and neck squamous cell carcinoma (SCC). The surgical excision of these cancers is challenging and often supplemented with radiotherapy. One novel concept to improve survival rates is to increase the susceptibility of SCCs to radiotherapy. ATR is a protein responsible for homologous recombination repair (HRR) within the FA pathway and has been identified as a possible target for cancer therapy. Inhibition of ATR induces subsequent DNA damage. The primary objective of this study was to identify whether the inhibition of ATR can be achieved by exposing SCCs to mild hyperthermia. This novel concept aims to improve survival rates of patients with SCCs who require surgical excision and subsequent radiotherapy. Method By using a HRR assay it was possible to examine levels of DNA repair in various SCC cell lines following the exposure to radiation. These were compared to cells treated with heat, an ATR inhibitor and a mixture of both to determine whether heat reduces HRR. Results Repeated HRR assay’s showed SCC’s treated with heat, ATR inhibitor and a mixture of both before exposure to radiation underwent significantly lower levels of HRR compared to cells within the control. Conclusions Reduced HRR following exposure to heat increases the susceptibility of SCCs to radiotherapy. It is hoped that in combination with the surgical excision, this study will help to improve the outcomes of patients with head and neck carcinomas by improving the efficacy of radiotherapy.