Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases ...classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.
We aimed to describe and analyse clinical features, characteristics, and adherence to surveillance guidelines in an Australian Birt-Hogg-Dubé syndrome (BHD) and hereditary leiomyomatosis and renal ...cell cancer (HLRCC) cohort.
All identified patients with a diagnosis of BHD or HLRCC at RBWH 01/01/2014-01/09/2019 were included (HREC/17/QRBW/276). All patients were initially assessed and counselled by a clinical geneticist and then referred to an adult nephrologist. Baseline and incidental clinical variables were extracted and analysed.
Fifty-seven patients were identified (28 BHD, 29 HLRCC) with a median age of 47 years. The median and cumulative follow-up were 1 and 99 years, respectively. Baseline renal MRI occurred in 40/57 patients, and 33/57 had regular MRI as per the national guidelines (eviQ). Of 18/57 without baseline imaging, nine were yet to have imaging, seven were lost follow-up, and two patients had logistic difficulties. RCC was diagnosed in 11/57 patients: two of 28 with BHD were diagnosed with RCC aged 73 and 77, both prior to commencement of surveillance. Nine of 29 patients with HLRCC were diagnosed with RCC (one of 29 during surveillance at 47 years of age) and eight of 29 prior to commencement of surveillance (11-55 years). Amongst BHD patients, cutaneous fibrofolliculomas were noted in 15 patients, lung cysts were detected in seven patients, spontaneous pneumothoraces in five patients, and parotid oncocytoma in two of 28. Amongst those with HLRCC, cutaneous leiomyomas were noted in 19/29, cutaneous leiomyosarcoma diagnosed in one of 29, and uterine fibroids in 13 female patients.
Evidence-based RCC screening in BHD and HLRCC cohort is feasible and able to identify incidental renal lesions. Multidisciplinary patient management enables expedited genetic counselling, diagnosis, longitudinal screening, and RCC management. The success of this clinical model warrants consideration of undertaking longitudinal screening of BHD and HLRCC patients by nephrologists.
Background Many families and individuals do not meet criteria for a known hereditary cancer syndrome but display unusual clusters of cancers. These families may carry pathogenic variants in cancer ...predisposition genes and be at higher risk for developing cancer. Methods This multi-centre prospective study recruited 195 cancer-affected participants suspected to have a hereditary cancer syndrome for whom previous clinical targeted genetic testing was either not informative or not available. To identify pathogenic disease-causing variants explaining participant presentation, germline whole-genome sequencing (WGS) and a comprehensive cancer virtual gene panel analysis were undertaken. Results Pathogenic variants consistent with the presenting cancer(s) were identified in 5.1% (10/195) of participants and pathogenic variants considered secondary findings with potential risk management implications were identified in another 9.7% (19/195) of participants. Health economic analysis estimated the marginal cost per case with an actionable variant was significantly lower for upfront WGS with virtual panel ($8744AUD) compared to standard testing followed by WGS ($24,894AUD). Financial analysis suggests that national adoption of diagnostic WGS testing would require a ninefold increase in government annual expenditure compared to conventional testing. Conclusions These findings make a case for replacing conventional testing with WGS to deliver clinically important benefits for cancer patients and families. The uptake of such an approach will depend on the perspectives of different payers on affordability. Keywords: Familial cancer, Genetics, Variants, Whole-genome sequencing, Diagnostic testing, Health economics
Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. Lynch syndrome specific tumor features were evaluated ...for their ability to support the ACMG/InSiGHT framework in classifying variants of uncertain clinical significance (VUS) in the MMR genes. Twenty-eight CRC or EC tumors from 25 VUS carriers (6x
, 9x
, 6x
, 4x
), underwent targeted tumor sequencing for the presence of microsatellite instability/MMR-deficiency (MSI-H/dMMR) status and identification of a somatic MMR mutation (second hit). Immunohistochemical testing for the presence of dMMR crypts/glands in normal tissue was also performed. The ACMG/InSiGHT framework reclassified 7/25 (28%) VUS to likely pathogenic (LP), three (12%) to benign/likely benign, and 15 (60%) VUS remained unchanged. For the seven re-classified LP variants comprising nine tumors, tumor sequencing confirmed MSI-H/dMMR (8/9, 88.9%) and a second hit (7/9, 77.8%). Of these LP reclassified variants where normal tissue was available, the presence of a dMMR crypt/gland was found in 2/4 (50%). Furthermore, a dMMR endometrial gland in a carrier of an
exon 1-6 duplication provides further support for an upgrade of this VUS to LP. Our study confirmed that identifying these Lynch syndrome features can improve MMR variant classification, enabling optimal clinical care.
MLH1 epimutation is characterised by constitutional monoallelic MLH1 promoter hypermethylation, which can cause colorectal cancer (CRC). Tumour molecular profiles of MLH1 epimutation CRCs were used ...to classify germline MLH1 promoter variants of uncertain significance and MLH1 methylated early-onset CRCs (EOCRCs). Genome-wide DNA methylation and somatic mutational profiles of tumours from two germline MLH1: c.-11C > T and one MLH1: c.-28A > G; 7C > T carriers and three MLH1 methylated EOCRCs (< 45 years) were compared with 38 reference CRCs. Methylation-sensitive droplet digital PCR (ddPCR) was used to detect mosaic MLH1 methylation in blood, normal mucosa and buccal DNA.
Genome-wide methylation-based Consensus Clustering identified four clusters where the tumour methylation profiles of germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs clustered with the constitutional MLH1 epimutation CRCs but not with the sporadic MLH1 methylated CRCs. Furthermore, monoallelic MLH1 methylation and APC promoter hypermethylation in tumour were observed in both MLH1 epimutation and germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs. Mosaic constitutional MLH1 methylation in MLH1: c.-11C > T carriers and 1 of 3 MLH1 methylated EOCRCs was identified by methylation-sensitive ddPCR.
Mosaic MLH1 epimutation underlies the CRC aetiology in MLH1: c.-11C > T germline carriers and a subset of MLH1 methylated EOCRCs. Tumour profiling and ultra-sensitive ddPCR methylation testing can be used to identify mosaic MLH1 epimutation carriers.
Abstract Mutations in dynamin-2 ( DNM2 ) cause autosomal dominant centronuclear myopathy (CNM). We report a series of 12 patients from eight families with CNM in whom we have identified a number of ...novel features that expand the reported clinicopathological phenotype. We identified two novel and five recurrent missense mutations in DNM2. Early clues to the diagnosis include relative weakness of neck flexors, external ophthalmoplegia and ptosis, although these are not present in all patients. Pes cavus was present in two patients, and in another two members of one family there was mild slowing of nerve conduction velocities. Whole-body MRI examination in two children and one adult revealed a similar pattern of involvement of selective muscles in head (lateral pterygoids), neck (extensors), trunk (paraspinal) and upper limbs (deep muscles of forearm). Findings in lower limbs and pelvic region were similar to that previously reported in adults with DNM2 mutations. Two patients presented with dystrophic changes as the predominant pathological feature on muscle biopsies; one of whom had a moderately raised creatine kinase, and both patients were initially diagnosed as congenital muscular dystrophy. DNM2 mutation analysis should be considered in patients with a suggestive clinical phenotype despite atypical histopathology, and MRI findings can be used to guide genetic testing. Subtle neuropathic features in some patients suggest an overlap with the DNM2 neuropathy phenotype. Missense mutations in the C-terminal region of the PH domain appear to be associated with a more severe clinical phenotype evident from infancy.
Background
We report a kindred referred for molecular investigation of severe hemophilia A in a young female in which extremely skewed X‐inactivation was observed in both the proband and her ...clinically normal mother.
Methods
Bidirectional Sanger sequencing of all F8 gene coding regions and exon/intron boundaries was undertaken. Methylation‐sensitive restriction enzymes were utilized to investigate skewed X‐inactivation using both a classical human androgen receptor (HUMARA) assay, and a novel method targeting differential methylation patterns in multiple informative X‐chromosome SNPs. Illumina Whole‐Genome Infinium microarray analysis was performed in the case‐parent trio (proband and both parents), and the proband's maternal grandmother.
Results
The proband was a cytogenetically normal female with severe hemophilia A resulting from a heterozygous F8 pathogenic variant inherited from her similarly affected father. No F8 mutation was identified in the proband's mother, however, both the proband and her mother both demonstrated completely skewed X‐chromosome inactivation (100%) in association with a previously unreported 2.3 Mb deletion at Xp22.2. At least three disease‐associated genes (FANCB, AP1S2, and PIGA) were contained within the deleted region.
Conclusions
We hypothesize that true “extreme” skewing of X‐inactivation (≥95%) is a rare occurrence, but when defined correctly there is a high probability of finding an X‐chromosome disease‐causing variant or larger deletion resulting in X‐inactivation through a survival disadvantage or cell lethal mechanism. We postulate that the 2.3 Mb Xp22.2 deletion identified in our kindred arose de novo in the proband's mother (on the grandfather's homolog), and produced extreme skewing of X‐inactivation via a “cell lethal” mechanism. We introduce a novel multitarget approach for X‐inactivation analysis using multiple informative differentially methylated SNPs, as an alternative to the classical single locus (HUMARA) method. We propose that for females with unexplained severe phenotypic expression of an X‐linked recessive disorder trio‐SNP microarray should be undertaken in combination with X‐inactivation analysis.
We report a kindred referred for molecular investigation of severe hemophilia A in a young female in which extremely skewed X‐inactivation was observed in both the proband and her clinically normal mother. No F8 mutation was identified in the proband's mother, however both the proband and her mother demonstrated completely skewed X‐chromosome inactivation in association with a previously unreported 2.3 Mb deletion at Xp22.2
We present prenatal and postnatal features of a patient with severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN). Mutation analysis confirmed the clinical diagnosis by ...detecting the FGFR3 Lys650Met mutation. This case, one of only six with molecular analysis reported in the literature, confirms the severe morbidity and adds to the reports with early mortality associated with SADDAN. Clinical-radiological characteristics of all reported cases of SADDAN are reviewed and discussed.
Most patients with Cowden syndrome have lesions in the gastrointestinal tract, characterized by multiple polyps of various histologic types in the large bowel, polyps in the upper gastrointestinal ...tract, and esophageal glycogenic acanthosis. However, pathologists are often unaware of the distinctive polyposis phenotype of Cowden syndrome. In this multicenter study, we report the spectrum of gastrointestinal manifestations in a series of 43 Cowden syndrome patients who had at least one endoscopy. The median age at the first endoscopy was 46 years and 58% were women. In 24 of 29 (83%) tested patients, a pathogenic germline mutation in PTEN was identified. The histology from 199 endoscopy procedures (67 upper gastrointestinal endoscopy and 132 colonoscopies) was reviewed. Hamartomatous polyps of the large bowel were the most common lesions, present in 85% of patients. Hamartomatous polyps showed varied histology, including lymphoid aggregates in 55% of patients, a lipomatous component in 52%, a ganglioneuromatous component in 52%, and a fibrous-rich component in 14%. Polyps with at least two different stromal components were found in 55% of patients. Inflammatory polyps were present in 21% of patients. Conventional adenomas and serrated polyps were identified in 48% and 62% of patients, respectively. In the upper gastrointestinal tract, the most common lesions were esophageal glycogenic acanthosis (37%), gastric hamartomatous polyps (47%), and duodenal hamartomatous polyps (20%). All patients with glycogenic acanthosis who had a colonoscopy had hamartomatous polyps of the large bowel. In five patients, the diagnosis of Cowden syndrome was established after the pathology report raised suspicion for the diagnosis. Pathologists who are aware of the characteristic admixture of lesions in Cowden syndrome can play an essential role in recommending referral to genetic counseling and gene testing. Early diagnosis of Cowden syndrome is important, as these patients and their relatives are at increased risk for developing multiple cancers.
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