Whole genome sequencing identifies a homozygous splicing variant in TDRKH segregating with non-obstructive azoospermia in an Iranian family.

Non-obstructive azoospermia (NOA) resulting from primary spermatogenic failure represents one of the most severe forms of male infertility, largely because therapeutic options are very limited. Beyond their diagnostic value, genetic tests for NOA also hold prognostic potential. Specifically, genetic diagnosis enables the establishment of genotype-testicular phenotype correlations, which, in some cases, provide a negative predictive value for testicular sperm extraction (TESE), thereby preventing unnecessary surgical procedures. In this study, we employed whole-genome sequencing (WGS) to investigate two generations of an Iranian family with NOA and identified a homozygous splicing variant in TDRKH (NM_001083965.2: c.562-2A>T). TDRKH encodes a conserved mitochondrial membrane-anchored factor essential for piRNA biogenesis in germ cells. In Tdrkh knockout mice, de-repression of retrotransposons in germ cells leads to spermatogenic arrest and male infertility. Previously, our team reported TDRKH involvement in human NOA cases through the investigation of a North African cohort. This current study marks the second report of TDRKH's role in NOA and human male infertility, underscoring the significance of the piRNA pathway in spermatogenesis. Furthermore, across both studies, we demonstrated that men carrying TDRKH variants, similar to knockout mice, exhibit complete spermatogenic arrest, correlating with failed testicular sperm retrieval.

Splicing analysis of 24 potential spliceogenic variants in MMR genes and clinical interpretation based on refined ACMG/AMP criteria.

Lynch syndrome (LS) is a common hereditary cancer syndrome caused by heterozygous germline pathogenic variants in DNA mismatch repair (MMR) genes. Splicing defect constitutes one of the major mechanisms for MMR gene inactivation. Using RT-PCR based RNA analysis, we investigated 24 potential spliceogenic variants in MMR genes and determined their pathogenicity based on refined splicing-related American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria. Aberrant transcripts were confirmed in 19 variants and 17 of which were classified as pathogenic including 11 located outside of canonical splice sites. Most of these variants were previously reported in LS patients without mRNA splicing assessment. Thus, our study provides crucial evidence for pathogenicity determination, allowing for appropriate clinical follow-up. We also found that computational predictions were globally well correlated with RNA analysis results and the use of both SPiP and SpliceAI software appeared more efficient for splicing defect prediction.

PMS2 or PMS2CL? Characterization of variants detected in the 3' of the PMS2 gene.

PMS2 germline pathogenic variants are one of the major causes for Lynch syndrome and constitutional mismatch repair deficiencies. Variant identification in the 3' region of this gene is complicated by the presence of the pseudogene PMS2CL which shares a high sequence homology with PMS2. Consequently, short-fragment screening strategies (NGS, Sanger) may fail to discriminate variant's gene localization. Using a comprehensive analysis strategy, we assessed 42 NGS-detected variants in 76 patients and found 32 localized on PMS2 while 6 on PMS2CL. Interestingly, four variants were detected in either of them in different patients. Clinical phenotype was well correlated to genotype, making it very helpful in variant assessment. Our findings emphasize the necessity of more specific complementary analyses to confirm the gene origin of each variant detected in different individuals in order to avoid variant misinterpretation. In addition, we characterized two PMS2 genomic alterations involving Alu-mediated tandem duplication and gene conversion. Those mechanisms seemed to be particularly favored in PMS2 which contribute to frequent genomic rearrangements in the 3' region of the gene.

Overview of the Genetic Causes of Hereditary Breast and Ovarian Cancer Syndrome in a Large French Patient Cohort.

The use of multigene panel testing for patients with a predisposition to Hereditary Breast and Ovarian Cancer syndrome (HBOC) is increasing as the identification of mutations is useful for diagnosis and disease management. Here, we conducted a retrospective analysis of BRCA1/2 and non-BRCA gene sequencing in 4630 French HBOC suspected patients. Patients were investigated using a germline cancer panel including the 13 genes defined by The French Genetic and Cancer Group (GGC)-Unicancer. In the patients analyzed, 528 pathogenic and likely pathogenic variants (P/LP) were identified, including BRCA1 (n = 203, 38%), BRCA2 (n = 198, 37%), PALB2 (n = 46, 9%), RAD51C (n = 36, 7%), TP53 (n = 16, 3%), and RAD51D (n = 13, 2%). In addition, 35 novel (P/LP) variants, according to our knowledge, were identified, and double mutations in two distinct genes were found in five patients. Interestingly, retesting a subset of BRCA1/2-negative individuals with an expanded panel produced clinically relevant results in 5% of cases. Additionally, combining in silico (splicing impact prediction tools) and in vitro analyses (RT-PCR and Sanger sequencing) highlighted the deleterious impact of four candidate variants on splicing and translation. Our results present an overview of pathogenic variations of HBOC genes in the southeast of France, emphasizing the clinical relevance of cDNA analysis and the importance of retesting BRCA-negative individuals with an expanded panel.

From variant of unknown significance to likely pathogenic: Characterization and pathogenicity determination of a large genomic deletion in the MLH1 gene.

BACKGROUND: The MLH1 gene is one of the DNA mismatch repair genes (MMR), implicated in Lynch syndrome (LS), an autosomal dominant hereditary tumor susceptibility disease. The advent of next-generation sequencing (NGS) technologies has accelerated the diagnosis of inherited diseases and increased the percentage of diagnosis of inherited cancers. However, some complex genomic alterations require the combination of several analytical strategies to allow correct biological interpretations. Here, we describe a novel MLH1 deletion and its pathogenicity determination in a patient suspected of LS. METHODS: The index case was a French 73-year-old man diagnosed with colorectal cancer displaying microsatellite instability and the loss of MLH1 and PMS2 expression. NGS analysis was used as the primary method for MMR genes screening. Long-range PCR and reverse transcriptase polymerase chain reaction (RT-PCR) were used for breakpoints and pathogenicity determinations. RESULTS: A large genomic deletion was detected which removed the last six nucleotides of MLH1 exon 11 together with a large part of intron 11. It was initially considered as a variant of unknown significance (VUS). Genomic breakpoints were subsequently characterized defining the deletion as c.1033_1039-248del. Further RNA analysis demonstrated that this variant activated a cryptic donor splice site at the 5' of the breakpoint, leading to a premature truncated protein: p.Thr345Alafs*13. CONCLUSION: Our finding suggested that although NGS technologies have increased variant detection yield, combined approaches were still needed for complex variant characterization and pathogenicity assessment.

A PMS2 non-canonical splicing site variant leads to aberrant splicing in a patient suspected for lynch syndrome.

The PMS2 gene is one of the DNA mismatch repair genes (MMR) implicated in Lynch syndrome (LS). A subset of PMS2 pathogenic variants (PVs) are splice variants mostly affecting canonical GT/AG splicing sequences. However, the majority of the intronic variants outside canonical splice sites remained as variants of unknown significance, even though some of them would alter the splicing process. In this report, we describe the analysis of such an intronic variant (c.251-5T > C) detected in an 82-year-old patient diagnosed with endometrial cancer displaying microsatellite instability and the loss of PMS2 expression displayed. RNA analysis demonstrated that this variant lead to the complete exon 4 skipping, resulting in the synthesis of a truncated protein. This finding shows the relevance of functional RNA analysis in the non-canonical intronic variant assessment and the importance of systematic evaluation of MSI/loss of expression of MMR genes for LS screening in patients with endometrial cancers.

Identification and Characterization of an Exonic Duplication in PALB2 in a Man with Synchronous Breast and Prostate Cancer.

PALB2 (partner and localizer of BRCA2), as indicated by its name, is a BRCA2-interacting protein that plays an important role in homologous recombination (HR) and DNA double-strand break (DSB) repair. While pathogenic variants of PALB2 have been well proven to confer an increased risk of breast cancer, data on its involvement in prostate cancer (PrC) have not been clearly demonstrated. We investigated, using targeted next generation sequencing (NGS), a 59-year-old Caucasian man who developed synchronous breast and prostate cancers. This genetic investigation allowed to identify an intragenic germline heterozygous duplication in PALB2, implicating intronic repetitive sequences spanning exon 11. This variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints have been identified and characterized at the nucleotide level (c.3114-811_3202-1756dup) using an approach based on walking PCR, long range PCR, and Sanger sequencing. RT-PCR using mRNA extracted from lymphocytes and followed by Sanger sequencing revealed a tandem duplication r.3114_3201dup; p.(Gly1068Glufs * 14). This duplication results in the synthesis of a truncated, and most-likely, non-functional protein. These findings expand the phenotypic spectrum of PALB2 variants and may improve the yield of genetic diagnoses in this field.

Postoperative Outcomes After Laparoscopic Liver Resections in Low and High-Volume Centers: A Multicentric Case-Matched Comparative Study.

BACKGROUND: Laparoscopic liver resection (LLR) is the gold standard for liver resections. Despite its feasibility and safety in high-volume centers (HVC), its performance is controversial in low-volume centers (LVCs). We aimed to evaluate the results of LLR performed in LVC. METHODS: Patients who underwent LLR between 2013 and 2019 in three LVCs were compared after case-matching with those in an HVC using the Institut Mutualiste Montsouris LLR Difficulty Score (IMMLDS). RESULTS: Seventy-six patients treated in three LVCs were matched to 152 in HVCs for age, body mass index, and resection type. The incidence of LLR significantly increased in LVCs over time (2013-2016 vs. 2017-2019) (21.2% vs. 39.3%; p = 0.002 and) while abdominal drainage rate decreased (77.4% vs. 51.1%; p = 0.003). In IMMLDS group I (60 vs. 120 patients), higher Pringle maneuver (43.3% vs. 2.5%; p < 0.0001), median blood loss (175 ml vs. 50 ml; p < 0.0001), abdominal drainage (58.3% vs. 6.6%; p < 0.0001), and conversion rate (8.3% vs. 1.6%, p = 0.04) were observed in LVCs. The overall postoperative morbidity was comparable (Clavien I-II: p = 0.54; Clavien > II: p = 0.71). In IMMLDS groups II-III, Pringle maneuver (56.5% vs. 3.1%; p < 0.0001), blood loss (350 ml vs. 175 ml; p = 0.02), and abdominal drainage (75% vs. 28.3%; p = 0.004) were different; however, postoperative morbidity was not. The surgical difficulty notwithstanding, length of stay (group I: p = 0.13; group II-III: p = 0.93) and R0 surgical margin (group I: p = 0.3; group II-III p = 0.39) were not different between LVCs and HVCs. CONCLUSIONS: LLR performed at an LVC can be feasible and safe with acceptable morbidity.

Identification and Characterization of New Alu Element Insertion in the BRCA1 Exon 14 Associated with Hereditary Breast and Ovarian Cancer.

Hereditary breast and ovarian cancer syndrome (HBOC) is an autosomal dominant cancer predisposition syndrome characterized by an increased risk of breast and ovarian cancers. Germline pathogenic variants in BRCA1 are found in about 7-10% of all familial breast cancers and 10% of ovarian cancers. Alu elements are the most abundant mobile DNA element in the human genome and are known to affect the human genome by different mechanisms leading to human disease. We report here the detection, by next-generation sequencing (NGS) analysis coupled with a suitable bioinformatics pipeline, of an AluYb8 element in exon 14 of the BRCA1 gene in a family with HBOC history first classified as BRCA-negative by Sanger sequencing and first NGS analysis. The c.4475_c.4476insAluYb8 mutation impacts splicing and induces the skipping of exon 14. As a result, the produced mRNA contains a premature stop, leading to the production of a short and likely non-functional protein (pAla1453Glyfs*10). Overall, our study allowed us to identify a novel pathogenic variant in BRCA1 and showed the importance of bioinformatics tool improvement and versioning.

Classification of 101 BRCA1 and BRCA2 variants of uncertain significance by cosegregation study: A powerful approach.

Up to 80% of BRCA1 and BRCA2 genetic variants remain of uncertain clinical significance (VUSs). Only variants classified as pathogenic or likely pathogenic can guide breast and ovarian cancer prevention measures and treatment by PARP inhibitors. We report the first results of the ongoing French national COVAR (cosegregation variant) study, the aim of which is to classify BRCA1/2 VUSs. The classification method was a multifactorial model combining different associations between VUSs and cancer, including cosegregation data. At this time, among the 653 variants selected, 101 (15%) distinct variants shared by 1,624 families were classified as pathogenic/likely pathogenic or benign/likely benign by the COVAR study. Sixty-six of the 101 (65%) variants classified by COVAR would have remained VUSs without cosegregation data. Of note, among the 34 variants classified as pathogenic by COVAR, 16 remained VUSs or likely pathogenic when following the ACMG/AMP variant classification guidelines. Although the initiation and organization of cosegregation analyses require a considerable effort, the growing number of available genetic tests results in an increasing number of families sharing a particular variant, and thereby increases the power of such analyses. Here we demonstrate that variant cosegregation analyses are a powerful tool for the classification of variants in the BRCA1/2 breast-ovarian cancer predisposition genes.

Pancreas-preserving management in reinterventions for severe pancreatic fistula after pancreatoduodenectomy: a systematic review.

BACKGROUND: Life-threatening postoperative pancreatic fistula (LTPOPF) is the most feared complication after pancreatoduodenectomy (PD). Although completion pancreatectomy (CP) is usually performed when radiological management fails, the associated morbidity and mortality rates remain high. Here, we reviewed pancreas-preserving alternatives to CP. METHODS: The PubMed database was systematically searched for publications between 1983 and 2014, describing pancreas-preserving surgical treatment of the pancreas remnant (PR) after reintervention in a context of post-PD LTPOPF. RESULTS: A total of 12 articles including 140 patients were reviewed. Six different types of pancreas-preserving treatment were described: external wirsungostomy, simple drainage of the PF, closure of pancreatic stump, internal wirsungostomy, partial CP, and salvage pancreatogastrostomy after major leakage of a pancreatojejunostomy. The overall median survival rate was 75 % but rose to 83 % when patients undergoing only surgical drainage of the fistula were excluded. The median complication rate was 75 %, and the median length of hospital stay was 41.5 days. Further reintervention was required for 25 % of the patients. The median incidence of late diabetes was 22.5 %. The incidence of exocrine insufficiency ranged from 0 to 100 % depending on the intervention. CONCLUSION: Pancreas-preserving surgical management of the PR after LTPOPF can be performed with acceptable mortality and morbidity. These data suggest that CP should have a more precisely specified role in the management algorithm and should not be performed systematically.

Hepatocellular carcinoma in situs ambiguus: CT findings of a rare disposition.

Heterotaxic disorders or situs ambiguus are uncommon anatomical variations constituted by a partial mirror-image disposition of intra thoracic and/or abdominal solid organs. These variations are challenging because rarely met in a surgeon's career, and because of the coexistence of numerous other anatomical variants, like ones related to the asymmetrical organs, causing difficulties when a surgical management is required. We report the case of a 57-year-old patient presenting liver cirrhosis in which regular follow-up discovered a hepatocellular carcinoma of the right part of the liver associated to numerous anatomical variations in the setting of a situs ambiguus. This patient was successfully treated by a sub-segmentectomy via a right sub-costal laparotomy. There were neither peroperative nor postoperative complications. This case emphasizes the technical difficulties faced, successfully managed thanks to a good preoperative screening, and allows us to review literature of such a rare and challenging situation.

Volumetric gain of the liver after major hepatectomy in obese patients: a case-matched study in 84 patients.

OBJECTIVE: The objective was to determine the liver volumetric recovering capacity and postoperative course after major hepatectomy in obese patients through a case-matched study. BACKGROUND: In literature, the impact of obesity on liver recovering has been analyzed only indirectly in terms of morbimortality but never through volumetric assessment. PATIENTS AND METHOD: Between 2005 and 2011, 42 patients with body mass index (BMI) 30 or higher (Ob group) underwent major hepatectomy and were matched with 42 patients with BMI 25 or lower (NonOb group) on the magnitude of resection (number of resected segments ±1, remnant liver volume to total liver volume, RLV/TLV, ±5%). The RLV was measured on computed tomographic slices preoperatively and postoperatively at 1 month (RLV-1M) for all patients and within 3 to 12 months in 42 paired patients (median = 6 months, RLV-6M). Considering hepatomegaly in Ob group, RLV was also normalized to body weight (RLVBWR). The liver volumetric gain was expressed as a relative increase [(RLV-1M - RLV)/RLV] or increase in RLVBWR. RESULTS: The Ob and NonOb groups were comparable regarding clinicopathological data, except for arterial hypertension (48% vs 19%; P = 0.005), mean steatosis (24% vs 10%; P = 0.03), and fibrosis incidence (33% vs 10%; P = 0.008). Ob group showed longer operative time and higher blood losses. There were no intergroup differences in liver failure (both 7.1%) and 90-day morbimortality. Despite comparable RLV/TLV (38.1% vs 37.7%; P = 0.13), the relative liver volumetric gain at 1 month was significantly lower in Ob group (+93% vs +115%; P = 0.002), as well as RLVBWR increase (+0.59% vs +0.79%; P < 0.001). The RLV-1M represented 66.2% of initial TLV in Ob group compared with 73.8% (P = 0.005) in NonOb group. This delay in relative volumetric gain persisted at 6 months (+105.4% vs +137.6%; P = 0.009), the RLV-6M representing 71.2% vs 82.4% of initial TLV (P = 0.014). CONCLUSIONS: In a methodologically robust trial in the first cohort reported up to date, the regenerative response in obese patients was comparatively slower based on their initial TLV or body weight.

Liver endometriosis presenting as a liver mass associated with high blood levels of tumoral biomarkers.

Endometriosis is a dissemination of endometrial-like tissue outside the uterine cavity, responsible for pain and impaired fertility in women of childbearing age. Although endometriosis generally occurs in the pelvis, it can be located further away. We describe the case of a 35-year-old woman who was admitted for further evaluation of a cystic mass of the liver that had invaded the right ventricle and caused pain. Serum levels of the tumor markers CA 125, CA 15-3 and CA 19-9 were elevated. The tumor was resected with a small part of the right ventricle free wall, the diaphragm and the left liver lobe. A histological analysis confirmed that the mass was a benign endometrial cyst. The postoperative course was uneventful and the patient remains asymptomatic with 5 year follow-up. A diagnosis of endometriosis should be considered for thoraco-abdominal cystic masses associated with menses-related pain in women of childbearing age.

Mechanisms of splenic hypertrophy following hepatic resection.

BACKGROUND: Following hepatic resection, liver regeneration has been associated with concurrent splenic hypertrophy. The mechanisms of this phenomenon are unknown, may be multiple and include: splanchnic sequestration caused by a reduction in the hepatic mass; hepatic growth factors that may indirectly act on the spleen, and the redistribution of the total reticuloendothelial system. METHODS: Seventy-five patients (40 males; median age: 60 years) who underwent minor (16%) or major (84%) hepatectomy between September 2004 and October 2009 were included. Prospective measurements of liver and spleen volumes were obtained preoperatively and postoperatively 1 month after hepatectomy using computed tomography (CT). The future remnant liver volume (RLV) was calculated on preoperative CT and the extent of resection was expressed as the RLV divided by total liver volume (TLV). Liver and spleen hypertrophy were expressed according to the absolute gain or relative increase in the initial volumes (%).The presence of fibrosis >F1, associated extrahepatic resection (except minor resections), and previous hepatectomy (major or minor) within 3 months represented exclusion criteria. RESULTS: Mean ± standard deviation (SD) liver volume at 1 month was higher than RLV (1187 ± 286 cm(3) versus 764 ± 421 cm(3) ; P < 0.001). Mean ± SD splenic volume increased from 252 ± 100 cm(3) preoperatively to 300 ± 111 cm(3) at 1 month (P < 0.001). Liver and splenic hypertrophy were significant after major hepatectomies (+100% and +26%, respectively; P < 0.001), but not after minor hepatectomies. Liver hypertrophy was inversely correlated to RLV/TLV (r = -0.687, P < 0.001). Splenic hypertrophy was not correlated to RLV/TLV. Liver and splenic hypertrophy were linearly correlated (r = 0.495, P < 0.001). Neoadjuvant chemotherapy (n = 37), preoperative portal vein embolization (n = 10) and postoperative complications (overall: n = 25; major: n = 10; infectious: n = 6) had no impact on hepatic or splenic hypertrophy. CONCLUSIONS: Splenic hypertrophy occurred after major hepatectomy, but was not correlated to the extent of resection, by contrast with liver hypertrophy. Nevertheless, there was a linear correlation between splenic and liver hypertrophy. This correlation suggests the hypothesis of a splenic action of hepatic growth factors or a redistribution of the total reticuloendothelial system rather than an effect of reduction of the portal bed or hepatic outflow.

Right hepatectomy with extra-hepatic vascular division prior to transection: intention-to-treat analysis of a standardized policy.

BACKGROUND: Right hepatectomy (RH) is the most common type of major hepatectomy and can be achieved without portal triad clamping (PTC) in non-cirrhotic liver. The present study reviews our standardized policy of performing RH without systematic PTC. METHODS: One hundred and eighty-one consecutive RH were performed in non-cirrhotic patients, with division of the right afferent and efferent blood vessels prior to transection, without systematically using PTC. Prospectively collected data were analysed, focusing on the following endpoints: need for salvage PTC, ischaemic time, blood loss and post-operative outcome. RESULTS: Extra-hepatic division of the right hepatic vessels was feasible in all patients, but was ineffective in 48 patients (26.5%) who required salvage PTC during transection. In those patients, the median ischaemic time was 20 min. The median blood loss was 500 ml (50-3000). Six patients (3.3%) experienced post-operative liver failure. Overall morbidity, severe morbidity and mortality were 42%, 12.1% and 1.6%, respectively, with peri-operative transfusion rate (16.6%) being the only factor associated with morbidity. DISCUSSION: By performing RH with extra-hepatic vascular division prior to transection, PTC can be safely avoided in the majority of patients.