A holistic approach to maximise diagnostic output in trio exome sequencing.

Introduction: Rare genetic diseases are a major cause for severe illness in children. Whole exome sequencing (WES) is a powerful tool for identifying genetic causes of rare diseases. For a better and faster assessment of the vast number of variants that are identified in the index patient in WES, parental sequencing can be applied ("trio WES"). Methods: We assessed the diagnostic rate of routine trio WES including analysis of copy number variants in 224 pediatric patients during an evaluation period of three years. Results: Trio WES provided a diagnosis in 67 (30%) of all 224 analysed children. The turnaround time of trio WES analysis has been reduced significantly from 41 days in 2019 to 23 days in 2021. Copy number variants could be identified to be causative in 10 cases (4.5%), underlying the importance of copy number variant analysis. Variants in three genes which were previously not associated with a clinical condition (GAD1, TMEM222 and ZNFX1) were identified using the matching tool GeneMatcher and were part of the first description of a new syndrome. Discussion: Trio WES has proven to have a high diagnostic yield and to shorten the process of identifying the correct diagnosis in paediatric patients. Re-evaluation of all 224 trio WES 1-3 years after initial analysis did not establish new diagnoses. Initiating (trio) WES as a first-tier diagnostics including copy number variant detection should be considered as early as possible, especially for children treated in ICU, if a monogenetic disease is suspected.

MiR-129-5p exerts Wnt signaling-dependent tumor-suppressive functions in hepatocellular carcinoma by directly targeting hepatoma-derived growth factor HDGF.

BACKGROUND: In hepatocellular carcinoma (HCC), histone deacetylases (HDACs) are frequently overexpressed. This results in chromatin compaction and silencing of tumor-relevant genes and microRNAs. Modulation of microRNA expression is a potential treatment option for HCC. Therefore, we aimed to characterize the epigenetically regulated miR-129-5p regarding its functional effects and target genes to understand its relevance for HCC tumorigenesis. METHODS: Global miRNA expression of HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B) and normal liver cell lines (THLE-2, THLE-3) was analyzed after HDAC inhibition by miRNA sequencing. An in vivo xenograft mouse model and in vitro assays were used to investigate tumor-relevant functional effects following miR-129-5p transfection of HCC cells. To validate hepatoma-derived growth factor (HDGF) as a direct target gene of miR-129-5p, luciferase reporter assays were performed. Survival data and HDGF expression were analyzed in public HCC datasets. After siRNA-mediated knockdown of HDGF, its cancer-related functions were examined. RESULTS: HDAC inhibition induced the expression of miR-129-5p. Transfection of miR-129-5p increased the apoptosis of HCC cells, decreased proliferation, migration and ERK signaling in vitro and inhibited tumor growth in vivo. Direct binding of miR-129-5p to the 3'UTR of HDGF via a noncanonical binding site was validated by luciferase reporter assays. HDGF knockdown reduced cell viability and migration and increased apoptosis in Wnt-inactive HCC cells. These in vitro results were in line with the analysis of public HCC datasets showing that HDGF overexpression correlated with a worse survival prognosis, primarily in Wnt-inactive HCCs. CONCLUSIONS: This study provides detailed insights into the regulatory network of the tumor-suppressive, epigenetically regulated miR-129-5p in HCC. Our results reveal for the first time that the therapeutic application of mir-129-5p may have significant implications for the personalized treatment of patients with Wnt-inactive, advanced HCC by directly regulating HDGF. Therefore, miR-129-5p is a promising candidate for a microRNA replacement therapy to prevent HCC progression and tumor metastasis.

Expanding the Phenotypic Spectrum of GPI Anchoring Deficiency Due to Biallelic Variants in GPAA1.

BACKGROUND AND OBJECTIVES: To expand the clinical knowledge of GPAA1-related glycosylphosphatidylinositol (GPI) deficiency. METHODS: An international case series of 7 patients with biallelic GPAA1 variants were identified. Clinical, biochemical, and neuroimaging data were collected for comparison. Where possible, GPI-anchored proteins were assessed using flow cytometry. RESULTS: Ten novel variants were identified in 7 patients. Flow cytometry samples of 3 available patients confirmed deficiency of several GPI-anchored proteins on leukocytes. Extensive phenotypic information was available for each patient. The majority experienced developmental delay, seizures, and hypotonia. Neuroimaging revealed cerebellar anomalies in the majority of the patients. Alkaline phosphatase was within the normal range in 5 individuals and low in 1 individual, as has been noted in other transamidase defects. We notably describe individuals either less affected or older than the ones published previously. DISCUSSION: Clinical features of the cases reported broaden the spectrum of the known phenotype of GPAA1-related GPI deficiency, while outlining the importance of using functional studies such as flow cytometry to aid in variant classification.

Plasma Metabolome Signature Indicative of BRCA1 Germline Status Independent of Cancer Incidence.

Individuals carrying a pathogenic germline variant in the breast cancer predisposition gene BRCA1 (gBRCA1+) are prone to developing breast cancer. Apart from its well-known role in DNA repair, BRCA1 has been shown to powerfully impact cellular metabolism. While, in general, metabolic reprogramming was named a hallmark of cancer, disrupted metabolism has also been suggested to drive cancer cell evolution and malignant transformation by critically altering microenvironmental tissue integrity. Systemic metabolic effects induced by germline variants in cancer predisposition genes have been demonstrated before. Whether or not systemic metabolic alterations exist in gBRCA1+ individuals independent of cancer incidence has not been investigated yet. We therefore profiled the plasma metabolome of 72 gBRCA1+ women and 72 age-matched female controls, none of whom (carriers and non-carriers) had a prior cancer diagnosis and all of whom were cancer-free during the follow-up period. We detected one single metabolite, pyruvate, and two metabolite ratios involving pyruvate, lactate, and a metabolite of yet unknown structure, significantly altered between the two cohorts. A machine learning signature of metabolite ratios was able to correctly distinguish between gBRCA1+ and controls in ~82%. The results of this study point to innate systemic metabolic differences in gBRCA1+ women independent of cancer incidence and raise the question as to whether or not constitutional alterations in energy metabolism may be involved in the etiology of BRCA1-associated breast cancer.

Psychological Distress and Coping Ability of Women at High Risk of Hereditary Breast and Ovarian Cancer before Undergoing Genetic Counseling-An Exploratory Study from Germany.

Carriers of pathogenic variants causing hereditary breast and ovarian cancer (HBOC) are confronted with a high risk to develop malignancies early in life. The present study aimed to determine the type of psychological distress and coping ability in women with a suspicion of HBOC. In particular, we were interested if the self-assessed genetic risk had an influence on health concerns and coping ability. Using a questionnaire established by the German HBOC Consortium, we investigated 255 women with breast cancer and 161 healthy women before they were seen for genetic counseling. The group of healthy women was divided into groups of high and low self-assessed risk. In our study, healthy women with a high self-assessed risk stated the highest stress level and worries about their health and future. A quarter of the women requested psychological support. Overall, only few women (4-11%) stated that they did not feel able to cope with the genetic test result. More women (11-23%, highest values in the low-risk group) worried about the coping ability of relatives. The results of our exploratory study demonstrate that the women, who presented at the Department of Human Genetics, Hanover Medical School, Germany were aware of their genetic risk and had severe concerns about their future health, but still felt able to cope with the genetic test result.

The identification of pathogenic variants in BRCA1/2 negative, high risk, hereditary breast and/or ovarian cancer patients: High frequency of FANCM pathogenic variants.

NGS-based multiple gene panel resequencing in combination with a high resolution CGH-array was used to identify genetic risk factors for hereditary breast and/or ovarian cancer in 237 high risk patients who were previously tested negative for pathogenic BRCA1/2 variants. All patients were screened for pathogenic variants in 94 different cancer predisposing genes. We identified 32 pathogenic variants in 14 different genes (ATM, BLM, BRCA1, CDH1, CHEK2, FANCG, FANCM, FH, HRAS, PALB2, PMS2, PTEN, RAD51C and NBN) in 30 patients (12.7%). Two pathogenic BRCA1 variants that were previously undetected due to less comprehensive and sensitive methods were found. Five pathogenic variants are novel, three of which occur in genes yet unrelated to hereditary breast and/or ovarian cancer (FANCG, FH and HRAS). In our cohort we discovered a remarkably high frequency of truncating variants in FANCM (2.1%), which has recently been suggested as a susceptibility gene for hereditary breast cancer. Two patients of our cohort carried two different pathogenic variants each and 10 other patients in whom a pathogenic variant was confirmed also harbored a variant of unknown significance in a breast and ovarian cancer susceptibility gene. We were able to identify pathogenic variants predisposing for tumor formation in 12.3% of BRCA1/2 negative breast and/or ovarian cancer patients.

12q14 microdeletion syndrome: A family with short stature and Silver-Russell syndrome (SRS)-like phenotype and review of the literature.

We report here on the first family with short stature and Silver-Russell-like phenotype due to a microdeletion in 12q14.3. The Netchine-Harbison clinical scoring system was used for the clinical diagnosis of Silver-Russell syndrome (SRS). The three affected first-degree relatives (index patient, mother and brother) presented with prenatal and postnatal growth retardation, feeding difficulties, a prominent forehead and a failure to thrive, but did not show relative macrocephaly. In addition, our index patient showed dysmorphic facial features, periodically increased sweating, and scoliosis. Learning problems and cardiac arrhythmia presented as additional features of her brother. Using high-resolution array-CGH, heterozygosity for a 1.67 Mb deletion in 12q14.3 was detected in the index patient. The heterozygous loss was confirmed by MLPA in the index patient and the other two affected family members. The deletion includes the genes HMGA2, LLPH, TMBIM4, IRAK3, HELB, GRIP1, and the pseudogene RPSAP52. We conclude from these results and from the data of other patients reported in the literature that haploinsufficiency of HMGA2 leads to the short stature in this family.