Merged testing for colorectal cancer syndromes and re-evaluation of genetic variants improve diagnostic yield: Results from a nationwide prospective cohort.

Approximately 5% of patients with colorectal cancer (CRC) have a Mendelian predisposition for the disease. Identification of the disease-causing genetic variant enables carrier testing and tailored cancer prevention within affected families. To determine the panorama and genetic variation of Mendelian CRC syndromes among referrals at the cancer genetics clinics in Sweden, 850 patients clinically selected for CRC genetic investigation were included in a prospective study that tested for all major hereditary polyposis and nonpolyposis CRC conditions. Genetically defined syndromes were diagnosed in 11% of the patients. Lynch syndrome was predominant (n = 73) followed by familial adenomatous polyposis (n = 12) and MUTYH-associated polyposis (n = 8); the latter of which two patients presented with CRC before polyposis was evident. One patient with a history of adolescent-onset CRC and polyposis had biallelic disease-causing variants diagnostic for constitutional mismatch repair deficiency syndrome. Post-study review of detected variants of unknown clinical significance (n = 129) resulted in the reclassification of variants as likely benign (n = 59) or as diagnostic for Lynch syndrome (n = 2). Our results reveal the panorama of Mendelian CRC syndromes at the cancer genetics clinics in Sweden and show that unified testing for polyposis and nonpolyposis CRC conditions as well as regular reexamination of sequence data improve the diagnostic yield.

Genetic anticipation in Swedish Lynch syndrome families.

Among hereditary colorectal cancer predisposing syndromes, Lynch syndrome (LS) caused by mutations in DNA mismatch repair genes MLH1, MSH2, MSH6 or PMS2 is the most common. Patients with LS have an increased risk of early onset colon and endometrial cancer, but also other tumors that generally have an earlier onset compared to the general population. However, age at first primary cancer varies within families and genetic anticipation, i.e. decreasing age at onset in successive generations, has been suggested in LS. Anticipation is a well-known phenomenon in e.g neurodegenerative diseases and several reports have studied anticipation in heritable cancer. The purpose of this study is to determine whether anticipation can be shown in a nationwide cohort of Swedish LS families referred to the regional departments of clinical genetics in Lund, Stockholm, Linköping, Uppsala and Umeå between the years 1990-2013. We analyzed a homogenous group of mutation carriers, utilizing information from both affected and non-affected family members. In total, 239 families with a mismatch repair gene mutation (96 MLH1 families, 90 MSH2 families including one family with an EPCAM-MSH2 deletion, 39 MSH6 families, 12 PMS2 families, and 2 MLH1+PMS2 families) comprising 1028 at-risk carriers were identified among the Swedish LS families, of which 1003 mutation carriers had available follow-up information and could be included in the study. Using a normal random effects model (NREM) we estimate a 2.1 year decrease in age of diagnosis per generation. An alternative analysis using a mixed-effects Cox proportional hazards model (COX-R) estimates a hazard ratio of exp(0.171), or about 1.19, for age of diagnosis between consecutive generations. LS-associated gene-specific anticipation effects are evident for MSH2 (2.6 years/generation for NREM and hazard ratio of 1.33 for COX-R) and PMS2 (7.3 years/generation and hazard ratio of 1.86). The estimated anticipation effects for MLH1 and MSH6 are smaller.

A retrospective study of extracolonic, non-endometrial cancer in Swedish Lynch syndrome families.

BACKGROUND: Lynch Syndrome is an autosomal dominant cancer syndrome caused by pathogenic germ-line variants in one of the DNA-mismatch-repair (MMR) genes MLH1, MSH2, MSH6 or PMS2. Carriers are predisposed to colorectal and endometrial cancer, but also other cancer types. The purpose of this retrospective study was to characterize the tumour spectrum of the Swedish Lynch syndrome families. METHODS: Data were obtained from genetically verified 235 Lynch families from five of the six health care regions in Sweden. The material was stratified for gender, primary cancer, age and mutated gene and the relative proportions of specific cancer types were compared to those in the general population. RESULTS: A total of 1053 family members had 1493 cancer diagnoses of which 1011 were colorectal or endometrial cancer. Individuals with pathogenic variants in MLH1 and MSH2 comprised 78% of the cohort. Among the 482 non-colorectal/non-endometrial cancer diagnoses, MSH2 carriers demonstrated a significantly increased proportion of urinary tract, gastric, small bowel, ovarian and non-melanoma skin cancer compared to the normal population. MLH1 carriers had an elevated proportion of gastrointestinal cancers (gastric, small bowel, pancreas), while MSH6 carriers had more ovarian cancer than expected. Gastric cancer was predominantly noted in older generations. CONCLUSION: Lynch syndrome confers an increased risk for multiple cancers other than colorectal and endometrial cancer. The proportions of other cancers vary between different MMR genes, with highest frequency in MSH2-carriers. Gender and age also affect the tumour spectrum, demonstrating the importance of additional environmental and constitutional parameters in determining the predisposition for different cancer types.

Muenke syndrome: An international multicenter natural history study.

Muenke syndrome is an autosomal dominant disorder characterized by coronal suture craniosynostosis, hearing loss, developmental delay, carpal, and calcaneal fusions, and behavioral differences. Reduced penetrance and variable expressivity contribute to the wide spectrum of clinical findings. Muenke syndrome constitutes the most common syndromic form of craniosynostosis, with an incidence of 1 in 30,000 births and is defined by the presence of the p.Pro250Arg mutation in FGFR3. Participants were recruited from international craniofacial surgery and genetic clinics. Affected individuals, parents, and their siblings, if available, were enrolled in the study if they had a p.Pro250Arg mutation in FGFR3. One hundred and six patients from 71 families participated in this study. In 51 informative probands, 33 cases (64.7%) were inherited. Eighty-five percent of the participants had craniosynostosis (16 of 103 did not have craniosynostosis), with 47.5% having bilateral and 28.2% with unilateral synostosis. Females and males were similarly affected with bicoronal craniosynostosis, 50% versus 44.4% (P = 0.84), respectively. Clefting was rare (1.1%). Hearing loss was identified in 70.8%, developmental delay in 66.3%, intellectual disability in 35.6%, attention deficit/hyperactivity disorder in 23.7%, and seizures in 20.2%. In patients with complete skeletal surveys (upper and lower extremity x-rays), 75% of individuals were found to have at least a single abnormal radiographical finding in addition to skull findings. This is the largest study of the natural history of Muenke syndrome, adding valuable clinical information to the care of these individuals including behavioral and cognitive impairment data, vision changes, and hearing loss.

Massive parallel sequencing-based non-invasive prenatal test (NIPT) identifies aberrations on chromosome 13.

OBJECTIVE: We report data of non-invasive prenatal testing (NIPT) at Uppsala University Hospital between 2017-2022. Furthermore, we illustrate the potential capacity of massive parallel sequencing-based NIPT beyond identification of common trisomies. METHODS: Maternal blood samples were analyzed using the Verifi NIPT or VeriSeq NIPT assays. Diagnostic testing, performed on amniotic fluid samples, included QF-PCR, microarray (SNP-array) and metaphase FISH. RESULTS: Among 4532 NIPT tests performed between 2017-2022, 125 samples (2.76%) showed increased risk for trisomies 13, 18, 21 and sex chromosome aneuploidy. For three patients with normal NIPT result further microarray indicated other types of chromosomal rearrangement which were not analyzed by NIPT. For another patient (case 1) the Verifi NIPT indicated trisomy 13. Fetal fraction (FF) was estimated to be 10%. Confirmatory microarray detected a segmental duplication on chromosome 13, as well as a terminal duplication and a terminal deletion on chromosome 10. A complex karyotype was observed in the fetus with metaphase FISH. In the second case the VeriSeq NIPT indicated trisomy 13. FF was estimated to be 11%. Confirmatory microarray detected a mosaicism of trisomy 13 in 30 % of cells. CONCLUSION: This study illustrates detection of peculiar abnormalities of chromosome 13 and supports potential to screen copy number variations with genome-wide NIPT.

A retrospective two centre study of Birt-Hogg-Dubé syndrome reveals a pathogenic founder mutation in FLCN in the Swedish population.

Birt-Hogg-Dube syndrome (BHDS) (MIM: 135150) is a rare autosomal dominant disorder with variable penetrance, caused by pathogenic variants in the FLCN gene. Only a few hundreds of families have so far been described in the literature. Patients with BHDS present with three distinct symptoms: fibrofolliculomas, pneumothorax due to lung cyst formation, and increased lifetime risk of kidney tumours. The aim of the current study was to estimate the incidence of BHDS in the Swedish population and further describe the clinical manifestations and their frequency. Splice variant c.779+1G>T was the most common pathogenic variant, found in 57% of the families, suggesting this may be a founder mutation in the Swedish population. This was further investigated using haplotype analysis in 50 families that shared a common haplotype. Moreover, according to gnomAD the carrier frequency of the c.779+1G>T variant has been estimated to be 1/3265 in the Swedish population, however our data suggest that the carrier frequency in the Swedish population may be significantly higher. These findings should raise awareness among physicians of different specialties to patients presenting with fibrofolliculomas, pneumothorax and/or kidney tumours. We also stress the importance of consensus recommendations regarding diagnosis and clinical management of this, not that uncommon, syndrome.

Simple structural chromosomal abnormalities in advanced stage of ovarian cancer.

BACKGROUND: Ovarian cancer represents the leading cause of death among patients with gynaecological cancer. The identification of chromosomal abnormalities is a useful strategy toward understanding tumourigenesis and specific chromosomal associations. Since single chromosomal changes might be primary events implicated in the initiation of the neoplastic process, the aim of the present study was to investigate the presence of simple structural chromosomal changes in ovarian cancer. MATERIALS AND METHODS: Reviewing on ascetic effusions samples cytogenetically studied by direct culture of tumour cells and a G-banding technique, two ovarian cancer cases were found which presented simple structural chromosomal abnormalities. RESULTS: The first case presented an abnormal clone of cells with an acquired pericentric inversion of chromosome 9, inv(9)(p11q13), as a sole anomaly. The second case presented simple chromosomal changes with involvement of the Xq23 chromosomal region, while a translocation t(X;11)(q23;q23) was also defined. CONCLUSIONS: The significance of the acquired pericentric inversion 9 in the development of the neoplastic process remains unknown. The chromosomal regions Xq23 and 11q23 need to be further investigated in association with clinicopathological parameters in ovarian cancer. The documentation of more ovarian cancer cases with simple chromosomal abnormalities is considered of major importance facilitating the identification of candidate genes involved in the neoplastic process. Improving the molecular understanding of ovarian cancer development and progression could facilitate the detection of specific tumour subtypes.

Corticotroph Pituitary Carcinoma in a Patient With Lynch Syndrome (LS) and Pituitary Tumors in a Nationwide LS Cohort.

Context: Lynch syndrome (LS) is a cancer-predisposing syndrome caused by germline mutations in genes involved in DNA mismatch repair (MMR). Patients are at high risk for several types of cancer, but pituitary tumors have not previously been reported. Case: A 51-year-old man with LS (MSH2 mutation) and a history of colon carcinoma presented with severe Cushing disease and a locally aggressive pituitary tumor. The tumor harbored a mutation consistent with the patient's germline mutation and displayed defect MMR function. Sixteen months later, the tumor had developed into a carcinoma with widespread liver metastases. The patient prompted us to perform a nationwide study in LS. Nationwide Study: A diagnosis consistent with a pituitary tumor was sought for in the Swedish National Patient Registry. In 910 patients with LS, representing all known cases in Sweden, another two clinically relevant pituitary tumors were found: an invasive nonsecreting macroadenoma and a microprolactinoma (i.e., in total three tumors vs. one expected). Conclusion: Germline mutations in MMR genes may contribute to the development and/or the clinical course of pituitary tumors. Because tumors with MMR mutations are susceptible to treatment with immune checkpoint inhibitors, we suggest to actively ask for a family history of LS in the workup of patients with aggressive pituitary tumors.

PHIP - a novel candidate breast cancer susceptibility locus on 6q14.1.

Most non-BRCA1/2 breast cancer families have no identified genetic cause. We used linkage and haplotype analyses in familial and sporadic breast cancer cases to identify a susceptibility locus on chromosome 6q. Two independent genome-wide linkage analysis studies suggested a 3 Mb locus on chromosome 6q and two unrelated Swedish families with a LOD >2 together seemed to share a haplotype in 6q14.1. We hypothesized that this region harbored a rare high-risk founder allele contributing to breast cancer in these two families. Sequencing of DNA and RNA from the two families did not detect any pathogenic mutations. Finally, 29 SNPs in the region were analyzed in 44,214 cases and 43,532 controls from BCAC, and the original haplotypes in the two families were suggested as low-risk alleles for European and Swedish women specifically. There was also some support for one additional independent moderate-risk allele in Swedish familial samples. The results were consistent with our previous findings in familial breast cancer and supported a breast cancer susceptibility locus at 6q14.1 around the PHIP gene.

Mismatch repair gene mutation spectrum in the Swedish Lynch syndrome population.

Lynch syndrome caused by constitutional mismatch­repair defects is one of the most common hereditary cancer syndromes with a high risk for colorectal, endometrial, ovarian and urothelial cancer. Lynch syndrome is caused by mutations in the mismatch repair (MMR) genes i.e., MLH1, MSH2, MSH6 and PMS2. After 20 years of genetic counseling and genetic testing for Lynch syndrome, we have compiled the mutation spectrum in Sweden with the aim to provide a population-based perspective on the contribution from the different MMR genes, the various types of mutations and the influence from founder mutations. Mutation data were collected on a national basis from all laboratories involved in genetic testing. Mutation analyses were performed using mainly Sanger sequencing and multiplex ligation-dependent probe amplification. A total of 201 unique disease-predisposing MMR gene mutations were identified in 369 Lynch syndrome families. These mutations affected MLH1 in 40%, MSH2 in 36%, MSH6 in 18% and PMS2 in 6% of the families. A large variety of mutations were identified with splice site mutations being the most common mutation type in MLH1 and frameshift mutations predominating in MSH2 and MSH6. Large deletions of one or several exons accounted for 21% of the mutations in MLH1 and MSH2 and 22% in PMS2, but were rare (4%) in MSH6. In 66% of the Lynch syndrome families the variants identified were private and the effect from founder mutations was limited and predominantly related to a Finnish founder mutation that accounted for 15% of the families with mutations in MLH1. In conclusion, the Swedish Lynch syndrome mutation spectrum is diverse with private MMR gene mutations in two-thirds of the families, has a significant contribution from internationally recognized mutations and a limited effect from founder mutations.

Protein expression in the brain of rat offspring in relation to prenatal caloric restriction.

OBJECTIVE: Intrauterine growth restriction (IUGR) has been associated with decreased supply of crucial substrates to the fetus and affects its growth and development by temporarily or permanently modifying gene expression and function. However, not all neonates born by calorie restricted mothers are IUGR and there are no reports regarding their brain protein expression vis-à-vis that of their IUGR siblings. Here, we investigated the expression of key proteins that regulate growth and development of the brain in non-IUGR newborn pups versus IUGR siblings and control pups. METHODS: Rat brain proteins were isolated from each group upon delivery and separated by two-dimensional gel electrophoresis (2-DE). RESULTS: 14-3-3 Protein, calreticulin, elongation factor, alpha-enolase, fascin, heat-shock protein HSP90 and pyruvate kinase isozymes were significantly increased (p < 0.05) in samples obtained from IUGR newborn pups compared to non-IUGR. Conversely, collapsin response mediator proteins, heat-shock70 and peroxiredoxin2 were decreased in IUGR group compared to non-IUGR. CONCLUSIONS: In our experimental study, IUGR pups showed an altered proteomic profile compared to their non-IUGR siblings and non-IUGR controls. Thus, not all offspring of calorie-restricted mothers become IUGR with the accompanying alterations in the expression of proteins. The differentially expressed proteins could modulate alterations in the energy balance, plasticity and maturation of the brain.

Screening for germline phosphatase and tensin homolog-mutations in suspected Cowden syndrome and Cowden syndrome-like families among uterine cancer patients.

Cowden syndrome (CS) is an autosomal dominant disorder characterized by multiple hamartomas in the breast, thyroid and endometrium, with a prevalence of 1 per 250,000. Females with CS have a 21-28% lifetime risk of developing uterine cancer. Germline mutations in the phosphatase and tensin homolog (PTEN) gene, a tumor suppressor gene, are responsible for 30-80% of CS cases. PTEN is a nine-exon gene, located on chromosome 10q23.3, which encodes the 403 amino acid PTEN protein. It negatively regulates the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway, affecting various cellular processes and signaling pathways. The present study examined whether PTEN mutations are present in CS-like families with uterine cancer (UC). UC patients underwent surgery at Karolinska University Hospital, Stockholm, Sweden (2008-2012). Pedigrees were analyzed and 54 unrelated CS-like families were identified. CS-like families were defined as having at least one occurrence of uterine cancer and one of breast cancer, as well as at least one additional Cowden-associated tumor (uterine, breast, thyroid, colon or kidney cancer) in the same individual or in first-degree relatives. Genomic DNA was amplified using polymerase chain reaction, and DNA sequencing analysis of all nine exons of the PTEN gene was conducted. No germline PTEN mutations or polymorphisms were identified. Germline PTEN mutations are rare in CS-like families with uterine cancer, therefore, genetic screening must be restricted to patients that meet the strict National Comprehensive Cancer Network criteria. Gynecologists must be aware of the CS criteria and identify potential cases of CS in females where uterine cancer is the sentinel cancer.

Whole-genome Linkage Analysis and Sequence Analysis of Candidate Loci in Familial Breast Cancer.

BACKGROUND: Known breast cancer-predisposing genes account for fewer than 25% of all familial breast cancer cases and further studies are required to find the remaining high- and moderate-risk genes. We set-out to couple linkage analysis using microsatellite marker data and sequence analysis of linked regions in 13 non-BRCA1/2 families in order to find novel susceptibility loci and high-penetrant genes. MATERIALS AND METHODS: Genotyping with 540 fluorescently-labeled microsatellite markers located on the 23 chromosomes at 7.25 cM resolution was used for primary linkage analysis and an additional 40 markers were used for fine-mapping of loci with a logarithm of odds (LOD) or heterogeneity LOD (HLOD) score greater than one. Whole-exome sequencing data of 28 members from all 13 families were used for the bioinformatics sequence analysis on the linked regions of these families. RESULTS: Linkage analysis identified three loci on chromosome 18q as a putative region of interest (overall LOD=1, HLOD=1.2). Sequencing analysis of the three linked regions on 18q and mutation prediction algorithms did reveal three probable damaging variants. CONCLUSION: Overall, our study identified three weakly linked loci on 18q and three probable damaging variants of interest in the 13 families with breast cancer.

A familial case of Muenke syndrome. Diverse expressivity of the FGFR3 Pro252Arg mutation--case report and review of the literature.

Muenke is a fibroblast growth factor receptor 3 (FGFR-3)-associated syndrome, which was first described in late 1990 s. Muenke syndrome is an autosomal dominant disorder characterized mainly by coronal suture craniosynostosis, hearing impairment and intellectual disability. The syndrome is defined molecularly by a unique point mutation c.749C > G in exon 7 of the FGFR3 gene which results to an amino acid substitution p.Pro250Arg of the protein product. Despite the fact that the mutation rate at this nucleotide is one of the most frequently described in human genome, few Muenke familial case reports are published in current literature. We describe individuals among three generations of a Greek family who are carriers of the same mutation. Medical record and physical examination of family members present a wide spectrum of clinical manifestations. In particular, a 38-year-old woman and her father appear milder clinical findings regarding craniofacial characteristics compared to her uncle and newborn female child. This familial case illustrates the variable expressivity of Muenke syndrome in association with an identical gene mutation.

Aberrant expression of collapsin response mediator proteins-1, -2 and -5 in the brain of intrauterine growth restricted rats.

Intrauterine growth restriction (IUGR) has been associated with increased perinatal morbidity, higher incidence of neurodevelopmental defects and increased risk for adult metabolic syndrome manifestations. Altered protein expression profiles associated with IUGR may be informative on the pathological mechanisms of this condition and might reveal potential markers for postnatal complications. We hypothesized that nutrient manipulation of the pregnant rat might influence the expression of important neurodevelopmental proteins in the resultant IUGR offspring. Therefore, we aimed to determine in newborn rat brain tissue the expression of collapsin response mediator proteins (CRMPs)-1, -2 and -5, commonly referred to as dihydropyrimidinase-related proteins (DPYLs) - playing a role in axon guidance, invasive growth and cell migration - and compare it to the corresponding expression in control rats. Two-dimensional electrophoresis and mass spectrometry, as well as Western blot analysis were employed in brain tissue from 24 IUGR newborn rats and 24 controls. With both methods, CRMP-1, CRMP-2 and CRMP-5 were decreased in the brains of the IUGR group as compared to the control group at the time of delivery. In conclusion, IUGR rat offspring are born with a decreased expression of CRMPs, suggesting that these proteins may be implicated in fetal stress-induced programming.

Detection of numerical abnormalities of chromosome 9 and p16/CDKN2A gene alterations in ovarian cancer with fish analysis.

BACKGROUND: The molecular events leading to the development of ovarian cancer are not well-established. Defects of the retinoblastoma protein (pRb)/cyclin-D1/p16 pathway have been shown to play a critical role in the development of human malignancies. In particular, the p16/cyclin-dependent kinase inhibitor 2A (CDKN2A) gene located on chromosomal region 9p21 frequently is altered in several types of cancer. MATERIALS AND METHODS: To investigate both the presence of numerical abnormalities of chromosome 9 and p16 gene alterations in ovarian cancer, we studied 28 cases by the fluorescence in situ hybridization (FISH) technique using a DNA p16 probe and an a-satellite probe specific for chromosome 9. RESULTS: Numerical abnormalities of chromosome 9 were found in all studied cases. Polysomy 9 was detected in 10 cases while monosomy 9 in seven cases. In 11 cases, there were two cell populations, one with polysomy 9 and the other with monosomy 9. In all cases, the p16 gene deletion was observed. Among them, 25 cases presented deletion of p16 gene in 21.43%-86.3% of the examined cells. Three cases carried deletion of the p16 gene in a lower proportion (12.04%-19.49%). In five cases with p16 gene deletion, homozygous deletion was detected. CONCLUSION: Numerical aberrations of chromosome 9 and p16 gene deletion are common findings in ovarian cancer. Data suggest that the p16 gene, located in the short arms of chromosome 9, may play a role in ovarian carcinogenesis. In addition, polysomy 9 could lead to activation of a number of oncogenes, thus participating in the neoplastic process in the ovaries.

Frontotemporal dementia, manifested as schizophrenia, with decreased heterochromatin on chromosome 1.

Introduction. Frontotemporal dementia is a disorder of complex etiology, with genetic components contributing to the disease. The aim of this report is to describe a young patient suffering from frontotemporal dementia, misdiagnosed as schizophrenia, related to a genetic defect on chromosome 1. Case Presentation. A 29-year-old female patient, previously diagnosed as having schizophrenia, was hospitalized with severe behavioural disturbances. She demonstrated severe sexual disinhibition, hyperphagia, lack of motivation, apathy, psychotic symptoms, suicidal thoughts, and cognitive deterioration. Focal atrophy of frontal and anterior temporal structures bilaterally was found on brain MRI, as well as bifrontal hypo perfusion of the brain on SPECT scan. The diagnosis of frontotemporal dementia was made clinically, according to Lund and Manchester groups and Neary diagnostic criteria. Chromosomal analysis was conducted and revealed decrease in length of heterochromatin on the long arm of chromosome 1 (46, XX, 1qh-). Parental karyotypes were normal. Discussion. Frontotemporal dementia, and particularly early-onset cases, can be often misdiagnosed as schizophrenia, with negative impact on case management. Genetic testing could be an aid to the correct diagnosis, which is crucial for optimal patient care.