Infantile-onset parkinsonism, dyskinesia, and developmental delay: do not forget polyglutamine defects!

We present the phenotype of an infant with the largest ATN1 CAG expansion reported to date (98 repeats). He presented at 4 months with developmental delay, poor eye contact, acquired microcephaly, failure to thrive. He progressively developed dystonia-parkinsonism with paroxysmal oromandibular and limbs dyskinesia and fatal outcome at 17 months. Cerebral MRI disclosed globus pallidus T2-WI hyperintensities and brain atrophy. Molecular analysis was performed post-mortem following the diagnosis of dentatorubral-pallidoluysian atrophy (DRPLA) in his symptomatic father. Polyglutamine expansion defects should be considered when neurodegenerative genetic disease is suspected even in infancy and parkinsonism can be a presentation of infantile-onset DRPLA.

MSH3: a confirmed predisposing gene for adenomatous polyposis.

BACKGROUND: The MSH3 gene is part of the DNA mismatch repair system, but has never been shown to be involved in Lynch syndrome. A first report of four patients from two families, bearing biallelic MSH3 germline variants, with a phenotype of attenuated colorectal adenomatous polyposis raised the question of its involvement in hereditary cancer predisposition. The patients' tumours exhibited elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), a hallmark of MSH3 deficiency. METHODS: We report five new unrelated patients with MSH3-associated polyposis. We describe their personal and familial history and study the EMAST phenotype in various normal and tumour samples, which are relevant findings based on the rarity of this polyposis subtype so far. RESULTS: All patients had attenuated colorectal adenomatous polyposis, with duodenal polyposis in two cases. Both women had breast carcinomas. EMAST phenotype was present at various levels in different samples of the five patients, confirming the MSH3 deficiency, with a gradient of instability in polyps depending on their degree of dysplasia. The negative EMAST phenotype ruled out the diagnosis of germline MSH3 deficiency for two patients: one homozygous for a benign variant and one with a monoallelic large deletion. CONCLUSION: This report lends further credence to biallelic MSH3 germline pathogenic variants being involved in colorectal and duodenal adenomatous polyposis. Large-scale studies may help clarify the tumour spectrum and associated risks. Ascertainment of EMAST may help with the interpretation of variants of unknown significance. We recommend adding MSH3 to dedicated diagnostic gene panels.

Description of Copy Number Variations in a Series of Children and Adolescents with FASD in Reunion Island.

BACKGROUND: Fetal Alcohol Spectrum Disorders (FASD) are the most common cause of neurocognitive impairment and social inadaptation, affecting 1 birth in 100. Despite the existence of precise diagnostic criteria, the diagnosis remains difficult, often confounded with other genetic syndromes or neurodevelopmental disorders. Since 2016, Reunion Island has been a pilot region for the identification, diagnosis, and care of FASD in France. OBJECTIVE: To evaluate the prevalence and the types of Copy Number Variations (CNV) in FASD patients. METHODS: A retrospective chart review of 101 patients diagnosed with FASD in the Reference Center for developmental anomalies and in the FASD Diagnostic Center of the University Hospital was performed. Records of all patients were reviewed to obtain their medical history, family history, clinical phenotype, and investigations, including genetic testing (CGH- or SNP-array). RESULTS: A rate of 20.8% (n = 21) of CNVs was found including 57% (12/21) of pathogenic variants and 29% (6/21) of variants of uncertain signification (VUS). CONCLUSION: A particularly high number of CNVs was found in children and adolescents with FASD. It reinforces the plea for a multidisciplinary approach for developmental disorders to explore both environmental factors, such as avoidable teratogens and intrinsic vulnerabilities, especially genetic determinants.

Clonal hematopoiesis driven by chromosome 1q/MDM4 trisomy defines a canonical route toward leukemia in Fanconi anemia.

Fanconi anemia (FA) patients experience chromosome instability, yielding hematopoietic stem/progenitor cell (HSPC) exhaustion and predisposition to poor-prognosis myeloid leukemia. Based on a longitudinal cohort of 335 patients, we performed clinical, genomic, and functional studies in 62 patients with clonal evolution. We found a unique pattern of somatic structural variants and mutations that shares features of BRCA-related cancers, the FA-hallmark being unbalanced, microhomology-mediated translocations driving copy-number alterations. Half the patients developed chromosome 1q gain, driving clonal hematopoiesis through MDM4 trisomy downmodulating p53 signaling later followed by secondary acute myeloid lukemia genomic alterations. Functionally, MDM4 triplication conferred greater fitness to murine and human primary FA HSPCs, rescued inflammation-mediated bone marrow failure, and drove clonal dominance in FA mouse models, while targeting MDM4 impaired leukemia cells in vitro and in vivo. Our results identify a linear route toward secondary leukemogenesis whereby early MDM4-driven downregulation of basal p53 activation plays a pivotal role, opening monitoring and therapeutic prospects.

Robust methylation-based classification of brain tumours using nanopore sequencing.

BACKGROUND: DNA methylation-based classification of cancer provides a comprehensive molecular approach to diagnose tumours. In fact, DNA methylation profiling of human brain tumours already profoundly impacts clinical neuro-oncology. However, current implementation using hybridisation microarrays is time consuming and costly. We recently reported on shallow nanopore whole-genome sequencing for rapid and cost-effective generation of genome-wide 5-methylcytosine profiles as input to supervised classification. Here, we demonstrate that this approach allows us to discriminate a wide spectrum of primary brain tumours. RESULTS: Using public reference data of 82 distinct tumour entities, we performed nanopore genome sequencing on 382 tissue samples covering 46 brain tumour (sub)types. Using bootstrap sampling in a cohort of 55 cases, we found that a minimum set of 1000 random CpG features is sufficient for high-confidence classification by ad hoc random forests. We implemented score recalibration as a confidence measure for interpretation in a clinical context and empirically determined a platform-specific threshold in a randomly sampled discovery cohort (N = 185). Applying this cut-off to an independent validation series (n = 184) yielded 148 classifiable cases (sensitivity 80.4%) and demonstrated 100% specificity. Cross-lab validation demonstrated robustness with concordant results across four laboratories in 10/11 (90.9%) cases. In a prospective benchmarking (N = 15), the median time to results was 21.1 h. CONCLUSIONS: In conclusion, nanopore sequencing allows robust and rapid methylation-based classification across the full spectrum of brain tumours. Platform-specific confidence scores facilitate clinical implementation for which prospective evaluation is warranted and ongoing.

Genotype-first approach to identify associations between CDH1 germline variants and cancer phenotypes: a multicentre study by the European Reference Network on Genetic Tumour Risk Syndromes.

BACKGROUND: Truncating pathogenic or likely pathogenic variants of CDH1 cause hereditary diffuse gastric cancer (HDGC), a tumour risk syndrome that predisposes carrier individuals to diffuse gastric and lobular breast cancer. Rare CDH1 missense variants are often classified as variants of unknown significance. We conducted a genotype-phenotype analysis in families carrying rare CDH1 variants, comparing cancer spectrum in carriers of pathogenic or likely pathogenic variants (PV/LPV; analysed jointly) or missense variants of unknown significance, assessing the frequency of families with lobular breast cancer among PV/LPV carrier families, and testing the performance of lobular breast cancer-expanded criteria for CDH1 testing. METHODS: This genotype-first study used retrospective diagnostic and clinical data from 854 carriers of 398 rare CDH1 variants and 1021 relatives, irrespective of HDGC clinical criteria, from 29 institutions in ten member-countries of the European Reference Network on Tumour Risk Syndromes (ERN GENTURIS). Data were collected from Oct 1, 2018, to Sept 20, 2022. Variants were classified by molecular type and clinical actionability with the American College of Medical Genetics and Association for Molecular Pathology CDH1 guidelines (version 2). Families were categorised by whether they fulfilled the 2015 and 2020 HDGC clinical criteria. Genotype-phenotype associations were analysed by Student's t test, Kruskal-Wallis, χ2, and multivariable logistic regression models. Performance of HDGC clinical criteria sets were assessed with an equivalence test and Youden index, and the areas under the receiver operating characteristic curves were compared by Z test. FINDINGS: From 1971 phenotypes (contributed by 854 probands and 1021 relatives aged 1-93 years), 460 had gastric and breast cancer histology available. CDH1 truncating PV/LPVs occurred in 176 (21%) of 854 families and missense variants of unknown significance in 169 (20%) families. Multivariable logistic regression comparing phenotypes occurring in families carrying PV/LPVs or missense variants of unknown significance showed that lobular breast cancer had the greatest positive association with the presence of PV/LPVs (odds ratio 12·39 [95% CI 2·66-57·74], p=0·0014), followed by diffuse gastric cancer (8·00 [2·18-29·39], p=0·0017) and gastric cancer (7·81 [2·03-29·96], p=0·0027). 136 (77%) of 176 families carrying PV/LPVs fulfilled the 2015 HDGC criteria. Of the remaining 40 (23%) families, who did not fulfil the 2015 criteria, 11 fulfilled the 2020 HDGC criteria, and 18 had lobular breast cancer only or lobular breast cancer and gastric cancer, but did not meet the 2020 criteria. No specific CDH1 variant was found to predispose individuals specifically to lobular breast cancer, although 12 (7%) of 176 PV/LPV carrier families had lobular breast cancer only. Addition of three new lobular breast cancer-centred criteria improved testing sensitivity while retaining high specificity. The probability of finding CDH1 PV/LPVs in patients fulfilling the lobular breast cancer-expanded criteria, compared with the 2020 criteria, increased significantly (AUC 0·92 vs 0·88; Z score 3·54; p=0·0004). INTERPRETATION: CDH1 PV/LPVs were positively associated with HDGC-related phenotypes (lobular breast cancer, diffuse gastric cancer, and gastric cancer), and no evidence for a positive association with these phenotypes was found for CDH1 missense variants of unknown significance. CDH1 PV/LPVs occurred often in families with lobular breast cancer who did not fulfil the 2020 HDGC criteria, supporting the expansion of lobular breast cancer-centred criteria. FUNDING: European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme.

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.

Recurrence of an early postzygotic rescue of an inherited unbalanced translocation resulting in mosaic segmental uniparental isodisomy of chromosome 11q in siblings.

Balanced translocations are associated with a risk of transmission of unbalanced chromosomal rearrangements in the offspring. Such inherited chromosomal abnormalities are typically non-mosaic as they are present in the germline. We report the recurrence in two siblings of a mosaicism for a chromosomal rearrangement inherited from their asymptomatic father who carried a balanced t(2;11)(q35;q25) translocation. Both siblings exhibited a similar phenotype including intellectual disability, dysmorphic features, kyphoscoliosis, and cervical spinal stenosis. Karyotyping, fluorescence in situ hybridization and SNP array analysis of blood lymphocytes of both siblings identified two cell lines: one carrying a 2q35q37.3 duplication and a 11q25qter deletion (~90% cells), and one carrying an 11q uniparental isodisomy of maternal origin (~10% cells). We hypothesize that these mosaics were related to a postzygotic rescue mechanism which unexpectedly recurred in both siblings.

Clinical and Genetic Features of Patients With Fanconi Anemia in Lebanon and Report on Novel Mutations in the FANCA and FANCG Genes.

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome and presents with cytopenias, characteristic physical features, increased chromosomal breaks, and a higher risk of malignancy. Genetic features of this disease vary among different ethnic groups. We aimed to identify the incidence, outcome, overall condition, and genetic features of patients affected with FA in Lebanon to optimize management, identify the most common genes, describe new mutations, and offer prenatal diagnosis and counseling to the affected families. Over a period of 17 years, 40 patients with FA were identified in 2 major diagnostic laboratories in Lebanon. Information was obtained on their clinical course and outcome from their primary physician. DNA was available in 20 patients and was studied for underlying mutations. FANCA seemed to be the most frequent genetic alteration and 2 novel mutations, one each in FANCA and FANCG, were identified. Nine patients developed various malignancies and died. This is the first study looking at clinical and genetic features of FA in Lebanon, and points to the need for establishing a national and regional registry for this condition.

Ovarian Clear Cell Carcinoma in Cowden Syndrome.

Cowden syndrome (CS) is an autosomal dominant mendelian disease related to germline pathogenic variants affecting the PTEN-gene. CS is characterized by macrocephaly, mucocutaneous lesions, and an increased risk of breast and thyroid cancers. Rare ovarian cancer cases (mostly embryonic tumors) associated with PTEN have been described in the literature, but no current CS guidelines are available for ovarian cancer risk management. We report on a woman diagnosed with ovarian clear cell carcinoma (OCCC) at 28 years of age. The patient displayed macrocephaly, trichilemmomas, oral papillomatosis, and acral keratosis. A family history of multiple cancer cases within the PTEN-related tumor spectrum was identified. In addition, PET scan and fine-needle biopsy results led to a diagnosis of thyroid follicular neoplasia. PTEN sequencing revealed that she carried a germline inherited pathogenic variant in exon 5 c.388C>T, p.(Arg130*) (NM_000314). Somatic mismatch repair immunohistochemistry analysis showed normal expression, and germline BRCA1/2 sequencing did not reveal pathogenic or likely pathogenic variants. An ovarian cell immunohistochemistry analysis reported total loss of PTEN expression, which strongly suggested the role of PTEN in the oncogenesis of this cancer. Hence, a total thyroid resection was performed instead of thyroid lobectomy and a risk-reducing bilateral mastectomy was discussed. Co-occurrence of this pathogenic germline mutation in PTEN in this patient, early development of OCCC at age 28 years, and total loss of PTEN expression in the tumor might support the involvement of PTEN in the carcinogenesis of her ovarian cancer. We describe a new ovarian cancer case with an atypical histologic type-clear cell carcinoma-in CS. This observation might be the first indication of the need to expand the PTEN-related tumor spectrum to incorporate OCCC. The CS diagnosis significantly changed the therapeutic outcome of this patient.

Optimization of the diagnosis of inherited colorectal cancer using NGS and capture of exonic and intronic sequences of panel genes.

We have developed and validated for the diagnosis of inherited colorectal cancer (CRC) a massive parallel sequencing strategy based on: (i) fast capture of exonic and intronic sequences from ten genes involved in Mendelian forms of CRC (MLH1, MSH2, MSH6, PMS2, APC, MUTYH, STK11, SMAD4, BMPR1A and PTEN); (ii) sequencing on MiSeq and NextSeq 500 Illumina platforms; (iii) a bioinformatic pipeline that includes BWA-Picard-GATK (Broad Institute) and CASAVA (Illumina) in parallel for mapping and variant calling, Alamut Batch (Interactive BioSoftware) for annotation, CANOES for CNV detection and finally, chimeric reads analysis for the detection of other types of structural variants (SVs). Analysis of 1644 new index cases allowed the identification of 323 patients with class 4 or 5 variants, corresponding to a 20% disease-causing variant detection rate. This rate reached 37% in patients with Lynch syndrome, suspected on the basis of tumour analyses. Thanks to this strategy, we detected overlapping phenotypes (e.g., MUTYH biallelic mutations mimicking Lynch syndrome), mosaic alterations and complex SVs such as a genomic deletion involving the last BMPR1A exons and PTEN, an Alu insertion within MSH2 exon 8 and a mosaic deletion of STK11 exons 3-10. This strategy allows, in a single step, detection of all types of CRC gene alterations including SVs and provides a high disease-causing variant detection rate, thus optimizing the diagnosis of inherited CRC.

Contribution of de novo and mosaic TP53 mutations to Li-Fraumeni syndrome.

BACKGROUND: Development of tumours such as adrenocortical carcinomas (ACC), choroid plexus tumours (CPT) or female breast cancers before age 31 or multiple primary cancers belonging to the Li-Fraumeni (LFS) spectrum is, independently of the familial history, highly suggestive of a germline TP53 mutation. The aim of this study was to determine the contribution of de novo and mosaic mutations to LFS. METHODS AND RESULTS: Among 328 unrelated patients harbouring a germline TP53 mutation identified by Sanger sequencing and/or QMPSF, we could show that the mutations had occurred de novo in 40 cases, without detectable parental age effect. Sanger sequencing revealed two mosaic mutations in a child with ACC and in an unaffected father of a child with medulloblastoma. Re-analysis of blood DNA by next-generation sequencing, performed at a depth above 500X, from 108 patients suggestive of LFS without detectable TP53 mutations, allowed us to identify 6 additional cases of mosaic TP53 mutations, in 2/49 children with ACC, 2/21 children with CPT, in 1/31 women with breast cancer before age 31 and in a patient who developed an osteosarcoma at age 12, a breast carcinoma and a breast sarcoma at age 35. CONCLUSIONS: This study performed on a large series of TP53 mutation carriers allows estimating the contribution to LFS of de novo mutations to at least 14% (48/336) and suggests that approximately one-fifth of these de novo mutations occur during embryonic development. Considering the medical impact of TP53 mutation identification, medical laboratories in charge of TP53 testing should ensure the detection of mosaic mutations.

IL28B polymorphism is associated with treatment response in patients with genotype 4 chronic hepatitis C.

BACKGROUND & AIMS: Polymorphisms in the region of the interleukin (IL)28B gene have been associated with pegylated-interferon (PEG-IFN) and ribavirin treatment response mainly in genotype 1 HCV infections. However, there are few data on HCV genotype 4 (HCV-4) infection. We evaluated, in a unique well-characterized cohort of HCV-4 patients, the association of IL28B polymorphism with response to treatment or liver disease severity. METHODS: This study included 164 HCV-4 patients from different ethnic groups (Egyptian, European, and Sub-Saharan African). Among these patients, 82 were studied for response and 160 for disease severity. Free DNA extracted from all the 164 patient's serum samples was analyzed by direct sequencing of the SNP rs12979860 of IL28B. Genetic and bio-clinical features from patients having sustained virological response (43 SVR patients) and from those who did not respond to treatment or had a relapse after the end of the treatment (39 NR patients) were compared. IL28B polymorphism was compared between the 78 patients with mild fibrosis (Metavir score F0-F1) and the 82 with advanced fibrosis (F2-F4). RESULTS: Our data showed a better treatment response rate of the C allele of the IL28B gene SNP rs12979860 (p=0.0008). The response rates were 81.8%, 46.5%, and 29.4% for genotype CC, CT, and TT, respectively. No significant relationship was found between rs12979860 and the severity of the disease. CONCLUSIONS: The SNP rs12979860 is strongly associated with SVR in patients infected with HCV-4, but not with liver disease severity. Analysis of IL28B genotype might be used to guide treatment for these patients.