Primary Hyperparathyroidism in Multiple Endocrine Neoplasia Type 2A in Denmark 1930­2021: A Nationwide Population-Based Retrospective Study

Studies of primary hyperparathyroidism (PHPT) in multiple endocrine neoplasia type 2A (MEN 2A) shows divergence in frequency, disease definition, reporting of clinical characteristics and traces of selection bias. This is a nationwide population-based retrospective study of PHPT in MEN 2A, suggesting a representative frequency, with complete reporting and a strict PHPT definition. The Danish MEN 2A cohort 1930-2021 was used. Of 204 MEN 2A cases, 16 had PHPT, resulting in a frequency of 8% (CI, 5-12). Age-related penetrance at 50 years was 8% (CI, 4-15). PHPT was seen in the American Thyroid Association moderate (ATA-MOD) and high (ATA-H) risk groups in 62% and 38% of carriers, respectively. Median age at PHPT diagnosis was 45 years (range, 21-79). A total of 75% were asymptomatic and 25% were symptomatic. Thirteen underwent parathyroid surgery, resulting in a cure of 69%, persistence in 8% and recurrence in 23%. In this first study with a clear PHPT definition and no selection bias, we found a lower frequency of PHPT and age-related penetrance, but a higher age at PHPT diagnosis than often cited. This might be affected by the Danish RET p.Cys611Tyr founder effect. Our study corroborates that PHPT in MEN 2A is often mild, asymptomatic and is associated with both ATA-MOD and ATA-H variants. Likelihood of cure is high, but recurrence is not infrequent and can occur decades after surgery.

Exploring associations between constipation, severity of neurofibromatosis type 1 and NF1 mutational spectrum.

Neurofibromatosis type 1 (NF1) is inherited in an autosomal dominant manner and is a rather common rare disease. Until recently, studies on gastrointestinal symptoms in patients with NF1 have been few and mostly described as case reports. In three previously published studies, the frequency of constipation in patients with NF1 has been found to be as high as 30%. In this study, associations between the frequency of constipation and NF1 disease severity and NF1 mutational spectrum were investigated. Among 277 patients with NF1, 49 had constipation. The highest rate of constipation was found among patients with a high perception of NF1 illness burden, and patients with constipation had a significantly higher NF1 illness burden when comparing the "not bothered" and the "very bothered" (p = 0.013). We found no significant association between constipation and the remaining measures on severity of NF1, nor between constipation and genetic variants. When observing the NF1 mutational spectrum, one variant (c.1013A>G (p.Asp338Gly/p.?) was identified in three patients with constipation of which two patients were related. The variant c.2970_2972delAAT (p.Met992del) associated with a mild NF1 phenotype was identified in two related patients with constipation. This study is the first to explore the association between symptoms of constipation, NF1 severity, and NF1 mutational spectrum. The results suggest an association between constipation and a high degree of illness burden. Awareness of this association among physicians could lead to more patients with NF1 being diagnosed with constipation. Constipation impacts on quality of life, hence a timely diagnosis and treatment will improve quality of life.

Long-term follow-up of RET Y791F carriers in Denmark 1994-2017: A National Cohort Study.

BACKGROUND AND OBJECTIVES: Recently, a comprehensive study presented evidence that a long-disputed REarranged during Transfection (RET) variant, RET Y791F, should be classified as nonpathogenic. In spite of this, several subsequently published papers, including the revised American Thyroid Association guidelines for medullary thyroid carcinoma, refer to the variant as pathogenic. This study presents data from a unique national Danish cohort of RET Y791F carriers who have been followed by watchful waiting instead of being subjected to early thyroidectomy, to determine if any carrier shows evidence of multiple endocrine neoplasia 2A (MEN2A) at long-term follow-up. METHODS: A national cohort of all patients tested for RET mutations in Denmark from September 1994 to October 2017 was searched for carriers of RET Y791F. Medical records and laboratory reports of carriers were reviewed for signs of MEN2A at latest follow-up (medullary thyroid carcinoma, primary hyperparathyroidism, pheochromocytoma, cutaneous lichen amyloidosis, or Hirschsprung's disease). RESULTS: In total, twenty RET Y791F-carriers were identified, none of whom showed any evidence of MEN2A, despite an age range from 7 to 87 years. CONCLUSIONS: Our national cohort study of all Danish RET Y791F carriers substantiates the claim that the RET Y791F variant is nonpathogenic.

Survival and Long-Term Biochemical Cure in Medullary Thyroid Carcinoma in Denmark 1997-2014: A Nationwide Study.

BACKGROUND: Survival of medullary thyroid carcinoma (MTC) subgroups in relation to the general population is poorly described. Data on the factors predicting long-term biochemical cure in MTC patients are nonexistent at a population level. A nationwide retrospective cohort study of MTC in Denmark from 1997 to 2014 was conducted, aiming to detect subgroups with survival similar to that of the general population and to identify prognostic factors for disease-specific survival and long-term biochemical cure. METHODS: The study included 220 patients identified from the nationwide Danish MTC cohort between 1997 and 2014. As a representative sample of the general population, a reference population matched 50:1 to the MTC cohort was used. RESULTS: Patients diagnosed with hereditary MTC by screening (hazard ratio [HR] = 1.5 [confidence interval (CI) 0.5-4.3]), patients without regional metastases (HR = 1.4 [CI 0.9-2.3]), and patients with stage I (HR = 1.3 [CI 0.6-3.1]), stage II (HR = 1.1 [CI 0.6-2.3]), and III (HR = 1.3 [CI 0.4-4.2]) disease had an overall survival similar to the reference population. On multivariate analysis, the presence of distant metastases (HR = 12.3 [CI 6.0-25.0]) predicted worse disease-specific survival, while the absence of regional lymph node metastases (odds ratio = 40.1 [CI 12.0-133.7]) was the only independent prognostic factor for long-term biochemical cure. CONCLUSIONS: Patients with hereditary MTC diagnosed by screening, patients without regional metastases, and patients with stages I, II, and III disease may have similar survival as the general population. The presence of distant metastases predicted worse disease-specific survival, while the absence of regional metastases predicted long-term biochemical cure.

Completeness of RET testing in patients with medullary thyroid carcinoma in Denmark 1997-2013: a nationwide study.

BACKGROUND: The completeness of REarranged during Transfection (RET) testing in patients with medullary thyroid carcinoma (MTC) was recently reported as 60%. However, the completeness on a population level is unknown. Similarly, it is unknown if the first Danish guidelines from 2002, recommending RET testing in all MTC patients, improved completeness in Denmark. We conducted a nationwide retrospective cohort study aiming to evaluate the completeness of RET testing in the Danish MTC cohort. Additionally, we aimed to assess the completeness before and after publication of the first Danish guidelines and characterize MTC patients who had not been tested. METHODS: The study included 200 patients identified from the nationwide Danish MTC cohort 1997-2013. To identify RET tested MTC patients before December 31, 2014, the MTC cohort was cross-checked with the nationwide Danish RET cohort 1994-2014. To characterize MTC patients who had not been RET tested, we reviewed their medical records and compared them with MTC patients who had been tested. RESULTS: Completeness of RET testing in the overall MTC cohort was 87% (95% CI: 0.81-0.91; 173/200). In the adjusted MTC cohort, after excluding patients diagnosed with hereditary MTC by screening, completeness was 83% (95% CI: 0.76-0.88; 131/158). Completeness was 88% (95% CI: 0.75-0.95; 42/48) and 81% (95% CI: 0.72-0.88) (89/110) before and after publication of the first Danish guidelines, respectively. Patients not RET tested had a higher median age at diagnosis compared to those RET tested. Median time to death was shorter in those not tested relative to those tested. CONCLUSION: The completeness of RET testing in MTC patients in Denmark seems to be higher than reported in other cohorts. No improvement in completeness was detected after publication of the first Danish guidelines. In addition, data indicate that advanced age and low life expectancy at MTC diagnosis may serve as prognostic indicators to identify patients having a higher likelihood of missing the compulsory RET test.

Replication of newly proposed TNM staging system for medullary thyroid carcinoma: a nationwide study.

A recent study proposed new TNM groupings for better survival discrimination among stage groups for medullary thyroid carcinoma (MTC) and validated these groupings in a population-based cohort in the United States. However, it is unknown how well the groupings perform in populations outside the United States. Consequently, we conducted the first population-based study aiming to evaluate if the recently proposed TNM groupings provide better survival discrimination than the current American Joint Committee on Cancer (AJCC) TNM staging system (seventh and eighth edition) in a nationwide MTC cohort outside the United States. This retrospective cohort study included 191 patients identified from the nationwide Danish MTC cohort between 1997 and 2014. In multivariate analysis, hazard ratios for overall survival under the current AJCC TNM staging system vs the proposed TNM groupings with stage I as reference were 1.32 (95% CI: 0.38-4.57) vs 3.04 (95% CI: 1.38-6.67) for stage II, 2.06 (95% CI: 0.45-9.39) vs 3.59 (95% CI: 1.61-8.03) for stage III and 5.87 (95% CI: 2.02-17.01) vs 59.26 (20.53-171.02) for stage IV. The newly proposed TNM groupings appear to provide better survival discrimination in the nationwide Danish MTC cohort than the current AJCC TNM staging. Adaption of the proposed TNM groupings by the current AJCC TNM staging system may potentially improve accurateness in survival discrimination. However, before such an adaption further population-based studies securing external validity are needed.

Incidence and prevalence of multiple endocrine neoplasia 2A in Denmark 1901-2014: a nationwide study.

BACKGROUND: The incidence and prevalence of multiple endocrine neoplasia 2A (MEN2A) have only been reported once in a nationwide setting. However, it is unclear whether the figures are representative of other populations, as the major component of the syndrome, hereditary medullary thyroid carcinoma (MTC), has been reported as rare in the same country. We conducted a nationwide retrospective cohort study of MEN2A in Denmark from 1901 to 2014, aiming to describe the incidence and prevalence. METHODS: This study included 250 unique MEN2A patients born or resident in Denmark before December 31, 2014. Patients were identified through the Danish REarranged during Transfection (RET) cohort, linkage of MEN2A pedigrees, the Danish MTC cohort, a nationwide collaboration of MEN2 centers, cross-checking of other relevant cohorts, and a systematic literature search. RESULTS: The incidence from 1971 to 2000 was 28 (95% CI: 21-37) per million live births per year. Incidence for the specific mutations or for the overall MEN2A group did not change significantly from 1901 to 2014 (P>0.05). Point prevalence at January 1, 2015, was 24 per million (95% CI: 20-28). CONCLUSION: The incidence and prevalence of MEN2A in Denmark seem higher than those reported in other countries. This is likely explained by the Danish C611Y founder effect. Also, our data indicate no significant change in MEN2A incidence during the last century.

Incidence and prevalence of sporadic and hereditary MTC in Denmark 1960-2014: a nationwide study.

Recent studies have shown a significant increase in the temporal trend of medullary thyroid carcinoma (MTC) incidence. However, it remains unknown to which extent sporadic medullary thyroid carcinoma (SMTC) and hereditary MTC (HMTC) affect the MTC incidence over time. We conducted a nationwide retrospective study using previously described RET and MTC cohorts combined with review of medical records, pedigree comparison and relevant nationwide registries. The study included 474 MTC patients diagnosed in Denmark between 1960 and 2014. In the nationwide period from 1997 to 2014, we recorded a mean age-standardized incidence of all MTC, SMTC and HMTC of 0.19, 0.13 and 0.06 per 100,000 per year, respectively. The average annual percentage change in incidence for all MTC, SMTC and HMTC were 1.0 (P = 0.542), 2.8 (P = 0.125) and -3.1 (P = 0.324), respectively. The corresponding figures for point prevalence at January 1, 2015 were 3.8, 2.5 and 1.3 per 100,000, respectively. The average annual percentage change in prevalence from 1998 to 2015 for all MTC, SMTC and HMTC was 2.8 (P < 0.001), 3.8 (P < 0.001) and 1.5 (P = 0.010), respectively. We found no significant change in the incidence of all MTC, SMTC and HMTC possibly due to our small sample size. However, due to an increasing trend in the incidence of all MTC and opposing trends of SMTC (increasing) and HMTC (decreasing) incidence, it seems plausible that an increase for all MTC seen by others may be driven by the SMTC group rather than the HMTC group.

Founder Effect of the RETC611Y Mutation in Multiple Endocrine Neoplasia 2A in Denmark: A Nationwide Study.

BACKGROUND: Multiple endocrine neoplasia (MEN) 2A and 2B are caused by REarranged during Transfection (RET) germline mutations. In a recent nationwide study, an unusually high prevalence (33%) of families with the C611Y mutation was reported, and it was hypothesized that this might be due to a founder effect. The first nationwide study of haplotypes in MEN2A families was conducted, with the aim of investigating the relatedness and occurrence of de novo mutations among Danish families carrying similar mutations. METHODS: The study included 21 apparently unrelated MEN2A families identified from a nationwide Danish RET cohort from 1994 to 2014. Twelve, two, two, three, and two families carried the C611Y, C618F, C618Y, C620R, and C634R mutations, respectively. Single nucleotide polymorphism chip data and identity by descent analysis were used to assess relatedness. RESULTS: A common founder mutation was found among all 12 C611Y families and between both C618Y families. No relatedness was identified in the remaining families. CONCLUSION: The data suggest that all families with the C611Y germline mutation in Denmark originate from a recent common ancestor, probably explaining the unusually high prevalence of this mutation. Additionally, the results indicate that the C611Y mutation rarely arises de novo, thus underlining the need for thorough multigenerational genetic work up in carriers of this mutation.

Evaluating the quality of Marfan genotype-phenotype correlations in existing FBN1 databases.

BACKGROUND: Genetic FBN1 testing is pivotal for confirming the clinical diagnosis of Marfan syndrome. In an effort to evaluate variant causality, FBN1 databases are often used. We evaluated the current databases regarding FBN1 variants and validated associated phenotype records with a new Marfan syndrome geno-phenotyping tool called the Marfan score. METHODS AND RESULTS: We evaluated four databases (UMD-FBN1, ClinVar, the Human Gene Mutation Database (HGMD), and Uniprot) containing 2,250 FBN1 variants supported by 4,904 records presented in 307 references. The Marfan score calculated for phenotype data from the records quantified variant associations with Marfan syndrome phenotype. We calculated a Marfan score for 1,283 variants, of which we confirmed the database diagnosis of Marfan syndrome in 77.1%. This represented only 35.8% of the total registered variants; 18.5-33.3% (UMD-FBN1 versus HGMD) of variants associated with Marfan syndrome in the databases could not be confirmed by the recorded phenotype. CONCLUSION: FBN1 databases can be imprecise and incomplete. Data should be used with caution when evaluating FBN1 variants. At present, the UMD-FBN1 database seems to be the biggest and best curated; therefore, it is the most comprehensive database. However, the need for better genotype-phenotype curated databases is evident, and we hereby present such a database.Genet Med advance online publication 01 December 2016.

Distribution of RET Mutations in Multiple Endocrine Neoplasia 2 in Denmark 1994-2014: A Nationwide Study.

BACKGROUND: Germline mutations of the REarranged during Transfection (RET) proto-oncogene cause multiple endocrine neoplasia 2 (MEN2). It is unclear whether the distribution of RET mutations varies among populations. The first nationwide study of the distribution of RET mutations was conducted, and the results were compared to those of other populations. METHODS: This retrospective cohort study included 1583 patients who underwent RET gene testing in one of three centers covering all of Denmark between September 1994 and December 2014. Primary testing method was Sanger sequencing, which included exons 8-11 and 13-16. Mutations were defined according to the ARUP database July 1, 2016. RESULTS: RET mutations were identified in 163 patients from 36 apparently unrelated families. Among the 36 families 13 (36.1%) carried mutations in codon 611, four (11.1%) in codon 618, three (8.3%) in codon 620, one (2.8%) in codon 631, six (16.7%) in codon 634, one (2.8%) in codon 790, one (2.8%) in codon 804, one (2.8%) in codon 852, one (2.8%) in codon 883, and five (13.9%) in codon 918. Among the 13 families with codon 611 mutations, 12 had the p.C611Y mutation. CONCLUSIONS: The distribution of RET mutations in Denmark appears to differ from that of other populations. Mutations in codon 611 were the most prevalent, followed by more frequently reported mutations. This might be due to a possible founder effect for the p.C611Y mutation. However, further studies are needed to find possible explanations for the skewed mutational spectrum in Denmark.

Molecular and biochemical investigations of patients with intermediate or severe hyperhomocysteinemia.

A discrepancy has been identified between numbers of expected and identified patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency. Patients homozygous for the frequent c.833T>C (p.I278T) are most often responsive to vitamin B6, and can present with a total-homocysteine (tHcy) <100 µM on a normal diet. In Denmark, patients with tHcy <100 µM are not routinely sequenced for CBS(2) mutations. This study investigated the prevalence of CBS mutations and the common methylenetetrahydrofolate reductase (MTHFR) c.677C>T polymorphism in patients with tHcy ≥ 50 µM and the association with clinical manifestations. We studied a cohort of patients with intermediate and severe hyperhomocysteinemia (tHcy ≥ 50 µM) determined between 1996 and 2011. Among the 413 eligible patients, 184 (45%) patients agreed to participate in the present follow-up study. A MTHFR(3)c.677TT genotype was found in 49% of the patients. Eight patients were found to have mutations in CBS(2). Of those, two were homozygous for c.833T>C (p.I278T), and four were compound heterozygous for c.833T>C. One c.833T>C (p.I278T) compound heterozygote was identified by lowering the threshold for sequencing from tHcy at 100 µM to 50 µM. The most prominent clinical presentation among patients with a CBS(2) mutation was thrombosis presenting at a median age of 25 years. In case of arterial or venous thrombosis without any explanation in individuals below 40 years, tHcy should be part of the thrombophilia screening. When tHcy is between 50 and 100 µM genotyping for the MTHFR(3) c.677TT is relevant, and when tHcy >100 µM CBS should be genotyped.

Difficulties in diagnosing Marfan syndrome using current FBN1 databases.

PURPOSE: The diagnostic criteria of Marfan syndrome (MFS) highlight the importance of a FBN1 mutation test in diagnosing MFS. As genetic sequencing becomes better, cheaper, and more accessible, the expected increase in the number of genetic tests will become evident, resulting in numerous genetic variants that need to be evaluated for disease-causing effects based on database information. The aim of this study was to evaluate genetic variants in four databases and review the relevant literature. METHODS: We assessed background data on 23 common variants registered in ESP6500 and classified as causing MFS in the Human Gene Mutation Database (HGMD). We evaluated data in four variant databases (HGMD, UMD-FBN1, ClinVar, and UniProt) according to the diagnostic criteria for MFS and compared the results with the classification of each variant in the four databases. RESULTS: None of the 23 variants was clearly associated with MFS, even though all classifications in the databases stated otherwise. CONCLUSION: A genetic diagnosis of MFS cannot reliably be based on current variant databases because they contain incorrectly interpreted conclusions on variants. Variants must be evaluated by time-consuming review of the background material in the databases and by combining these data with expert knowledge on MFS. This is a major problem because we expect even more genetic test results in the near future as a result of the reduced cost and process time for next-generation sequencing.Genet Med 18 1, 98-102.

Prevalence, incidence, and age at diagnosis in Marfan Syndrome.

BACKGROUND: Marfan syndrome is a genetic disorder with considerable morbidity and mortality. Presently, clinicians use the 2010 revised Ghent nosology, which includes optional genetic sequencing of the FBN1 gene, to diagnose patients. So far, only a few studies based on older diagnostic criteria have reported a wide range of prevalence and incidence. Our aim was to study prevalence, incidence, and age at diagnosis in patients with Marfan syndrome. METHOD: Using unique Danish patient-registries, we identified all possible Marfan syndrome patients recorded by the Danish healthcare system (1977-2014). Following, we confirmed or rejected the diagnosis according to the 2010 revised Ghent nosology. RESULTS: We identified a total of 1628 persons with possible Marfan syndrome. We confirmed the diagnosis in 412, whereof 46 were deceased, yielding a maximum prevalence of 6.5/100,000 at the end of 2014. The annual median incidence was 0.19/100,000 (range: 0.0-0.7) which increased significantly with an incidence rate ratio of 1.03 (95% CI: 1.02-1.04, p < 0.001). We found a median age at diagnose of 19.0 years (range: 0.0-74). The age at diagnosis increased during the study period, uninfluenced by the changes in diagnostic criteria. We found no gender differences. CONCLUSION: The increasing prevalence of Marfan syndrome during the study period is possibly due to build-up of a registry. Since early diagnosis is essential in preventing aortic events, diagnosing Marfan syndrome remains a task for both pediatricians and physicians caring for adults.

An unusual case of an ACTH-secreting macroadenoma with a germline variant in the aryl hydrocarbon receptor-interacting protein (AIP) gene.

UNLABELLED: A patient of Cushing's disease (CD) characterized by a large tumor and only subtle symptoms of hormonal hypersecretion was examined. The patient had a germline variant in the aryl hydrocarbon receptor-interacting protein (AIP) gene. A 50-year-old male presenting with headache was diagnosed with a large pituitary tumor by magnetic resonance imaging (MRI). His visual fields were intact and he exhibited no features of CD. Owing to an exuberant response to synacthen, an overnight dexamethasone suppression test was performed revealing inadequate suppression of plasma cortisol (419 nmol/l). Owing to tumor growth and visual field impairment, he underwent transsphenoidal surgery and developed hypocortisolemia. The pathology specimen revealed a sparsely granulated corticotrope adenoma. Postoperative MRI showed a large tumor remnant. The patient developed skin hyperpigmentation and a synacthen test demonstrated high basal and stimulated cortisol levels; an overnight dexamethasone suppression test showed no suppression (791 nmol/l) and elevated plasma ACTH levels (135 ng/l). A transcranial operation was performed followed by radiotherapy. Two months after radiotherapy, he developed secondary adrenocortical failure. Genetic testing revealed an AIP variant of unknown significance (p.R16H) without loss of the normal AIP allele in the tumor. A literature review showed ten CD patients with AIP gene variants, of whom five (including our case) were p.R16H. CD is occasionally dominated by pituitary tumor growth rather than symptoms of hypersecretion. The particular AIP gene variant identified in our patient is shared by four other reported cases of CD. Future studies are needed to assess whether the reported AIP gene variant is more than just coincidental. LEARNING POINTS: CD is occasionally dominated by pituitary tumor growth rather than symptoms of hypersecretion.Resolution of both tumor remnant and hormonal hypersecretion may occur within 2 months after postoperative radiotherapy.The particular AIP gene variant identified in our patient is shared by four other reported cases of CD.

[Genetic screening for mutations enables early diagnosis of pituitary adenomas]. / Genetisk screening for mutation muliggør tidlig diagnostik af hypofyseadenomer.

Mutations in the aryl hydrocarbon receptor interaction protein gene (AIP) occur in familial pituitary adenomas as an autosomal dominant inheritance with a 15-30% penetrance. The AIP mutation-associated pituitary adenomas are generally large, the onset of disease is early and treatment failure frequent. Genetic screening is also offered in Denmark and enables the detection of mutation carriers, and thus early diagnosis and treatment. Mutations have been recorded among Danish patients and their families.

Leiden Open Variation Database of the MUTYH gene.

The MUTYH gene encodes a DNA glycosylase involved in base excision repair (BER). Biallelic pathogenic MUTYH variants have been associated with colorectal polyposis and cancer. The pathogenicity of a few variants is beyond doubt, including c.536A4G/p.Tyr179Cys and c.1187G4A/p.Gly396Asp (previously c.494A4G/p.Tyr165Cys and c.1145G4A/p.Gly382Asp).However, for a substantial fraction of the detected variants, the clinical significance remains uncertain,compromising molecular diagnostics and thereby genetic counseling. We have established an interactive MUTYH gene sequence variant database (www.lovd.nl/MUTYH) with the aim of collecting and sharing MUTYH genotype and phenotype data worldwide. To support standard variant description, we chose NM_001128425.1 as the reference sequence. The database includes records with variants per individual, linked to available phenotype and geographic origin data as well as records with in vitro functional and in silico test data. As of April 2010, the database contains 1968 published and 423 unpublished submitted entries, and 230 and 61 unique variants,respectively. This open-access repository allows all involved to quickly share all variants encountered and communicate potential consequences, which will be especially useful to classify variants of uncertain significance.

A revisit to the natural history of homocystinuria due to cystathionine beta-synthase deficiency.

We review the evidence that in Denmark and probably certain other European countries the number of individuals identified with homocystinuria due to homozygosity for the widespread c.833T>C (p.I278T) mutation in the gene that encodes cystathionine beta-synthase (CBS) falls far short of the number of such individuals expected on the basis of the heterozygote frequency for this mutation found by molecular screening. We conclude that the predominant portion of such homozygotes may be clinically unaffected, or may be ascertained for thromboembolic events occurring no sooner than the third decade of life. If so, there was significant ascertainment bias in the time-to-event curves previously published describing the natural history of untreated CBS deficiency Mudd et al. and these curves should be used with care.

Alternative splicing in colon, bladder, and prostate cancer identified by exon array analysis.

Alternative splicing enhances proteome diversity and modulates cancer-associated proteins. To identify tissue- and tumor-specific alternative splicing, we used the GeneChip Human Exon 1.0 ST Array to measure whole-genome exon expression in 102 normal and cancer tissue samples of different stages from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder, and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by RT-PCR and sequencing. Furthermore 23, 19, and 18 candidate tumor-specific splicing alterations in colon, bladder, and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2, PIK4CB, TPM1, and VCL). The validated tumor-specific splicing alterations were highly consistent, enabling clear separation of normal and cancer samples and in some cases even of different tumor stages. A subset of the tumor-specific splicing alterations (ACTN1, CALD1, and VCL) was found in all three organs and may represent general cancer-related splicing events. In silico protein predictions suggest that the identified cancer-specific splice variants encode proteins with potentially altered functions, indicating that they may be involved in pathogenesis and hence represent novel therapeutic targets. In conclusion, we identified and validated alternative splicing between normal tissue samples from colon, bladder, and prostate in addition to cancer-specific splicing events in colon, bladder, and prostate cancer that may have diagnostic and prognostic implications.