A founder COL4A4 pathogenic variant resulting in autosomal recessive Alport syndrome accounts for most genetic kidney failure in Romani people.

Introduction: Romani people have a high prevalence of kidney failure. This study examined a Romani cohort for pathogenic variants in the COL4A3, COL4A4, and COL4A5 genes that are affected in Alport syndrome (AS), a common cause of genetic kidney disease, characterized by hematuria, proteinuria, end-stage kidney failure, hearing loss, and eye anomalies. Materials and methods: The study included 57 Romani from different families with clinical features that suggested AS who underwent next-generation sequencing (NGS) of the COL4A3, COL4A4, and COL4A5 genes, and 83 family members. Results: In total, 27 Romani (19%) had autosomal recessive AS caused by a homozygous pathogenic c.1598G>A, p.Gly533Asp variant in COL4A4 (n = 20) or a homozygous c.415G>C, p.Gly139Arg variant in COL4A3 (n = 7). For p.Gly533Asp, 12 (80%) had macroscopic hematuria, 12 (63%) developed end-stage kidney failure at a median age of 22 years, and 13 (67%) had hearing loss. For p.Gly139Arg, none had macroscopic hematuria (p = 0.023), three (50%) had end-stage kidney failure by a median age of 42 years (p = 0.653), and five (83%) had hearing loss (p = 0.367). The p.Gly533Asp variant was associated with a more severe phenotype than p.Gly139Arg, with an earlier age at end-stage kidney failure and more macroscopic hematuria. Microscopic hematuria was very common in heterozygotes with both p.Gly533Asp (91%) and p.Gly139Arg (92%). Conclusion: These two founder variants contribute to the high prevalence of kidney failure in Czech Romani. The estimated population frequency of autosomal recessive AS from these variants and consanguinity by descent is at least 1:11,000 in Czech Romani. This corresponds to a population frequency of autosomal dominant AS from these two variants alone of 1%. Romani with persistent hematuria should be offered genetic testing.

Guidelines for Genetic Testing and Management of Alport Syndrome.

Genetic testing for pathogenic COL4A3-5 variants is usually undertaken to investigate the cause of persistent hematuria, especially with a family history of hematuria or kidney function impairment. Alport syndrome experts now advocate genetic testing for persistent hematuria, even when a heterozygous pathogenic COL4A3 or COL4A4 is suspected, and cascade testing of their first-degree family members because of their risk of impaired kidney function. The experts recommend too that COL4A3 or COL4A4 heterozygotes do not act as kidney donors. Testing for variants in the COL4A3-COL4A5 genes should also be performed for persistent proteinuria and steroid-resistant nephrotic syndrome due to suspected inherited FSGS and for familial IgA glomerulonephritis and kidney failure of unknown cause.

Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome: refining the ACMG criteria.

The recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3 and COL4A4 genes beyond the classical Alport phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3-5). It identified 'mutational hotspots' (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that 'well-established' functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3 and COL4A4 variants were common 'incidental' findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3-COL4A5 genes remains a challenge.

Moderate sensorineural hearing loss is typical for DFNB16 caused by various types of mutations affecting the STRC gene.

INTRODUCTION: Hearing loss is the most frequent sensory disorder and is genetically extremely heterogeneous. By far the most frequent cause of nonsyndromic autosomal recessive hearing loss (AR-NSHL) are biallelic pathogenic mutations in the GJB2 gene causing DFNB1. The worldwide search for the second most common type of AR-NSHL took almost two decades. Recently reported alterations (mostly deletions) of the STRC gene, also named DFNB16, seem to be the second most frequent cause of AR-NSHL. Genetic testing of STRC is very challenging due to the highly homologous pseudogene. Anecdotal evidence from single patients shows that STRC mutations have their typical audiological findings and patients usually have moderate hearing loss. The aim of this study is to discover if audiological findings in patients with biallelic pathogenic mutations affecting STRC have the characteristic features and shape of audiological curves and if there are genotype/phenotype correlations in relation to various types of STRC mutations. METHODS: Eleven hearing loss patients with pathogenic mutations on both alleles of the STRC gene were detected during routine genetic examination of AR-NSHL patients. Audiological examination consisted of pure tone audiometry, stapedial reflexes, tympanometry and otoacoustic emission tests. RESULTS: The threshold of pure tone average (PTA) was 46 dB and otoacoustic emissions were not detectable in these DFNB16 patients. All patients were without vestibular irritation or asymmetry. CONCLUSION: Moderate sensorineural hearing loss is typical for DFNB16-associated hearing loss and there are no significant differences in audiological phenotypes among different types of mutations affecting STRC.

Genetic Causes of Rare Pediatric Ovarian Tumors.

BACKGROUND: Ovarian tumors in childhood and adolescence are distinguished from those that arise in adulthood by their histological subtype. These tumors may arise as the first manifestation of a cancer predisposition syndrome. Correct diagnosis of the syndrome may offer the possibility of surveillance for other members of the patients family. PURPOSE: To summarize current knowledge about paediatric ovarian tumors that may be associated with genetically defined cancer syndromes. Juvenile granulosa cell tumors occur in those with Ollier disease and Maffucci syndrome; they are caused by postzygotic IDH1 and IDH2 gene mutations. Sertoli-Leydig cell tumors usually arise in association with DICER1 syndrome, which is caused by germline DICER1 gene mutations. Sex cord tumors with annular tubules and Sertoli cell tumors may arise in patients with Peutz-Jeghers syndrome; this syndrome is caused by germline STK11 gene mutations. The majority of germ cell tumors develop in the context of gonadal dysgenesis. In XY gonadal dysgenesis, the presence of a Y chromosome material renders the patient at increased risk for developing gonadal malignancy. Characteristically, these patients develop gonadoblastoma, which has the potential to evolve into dysgerminoma and exhibit malignant behavior. Sex-chromosome aneuploidy syndromes or mutations in genes involved in gonadal development and differentiation may cause gonadal dysgenesis. Small cell carcinoma of the ovary of a hypercalcaemic type is usually caused by loss-of-function mutations in the SMARCA4 gene. CONCLUSION: Ovarian tumors are uncommon during childhood and adolescence. It is always necessary to consider gonadal dysgenesis or any of the inherited cancer syndromes. These patients require interdisciplinary care, careful noting of personal and family history, precise clinical examination, laboratory testing, and differential diagnosis by a clinician with a good knowledge of genetic syndromes. Expert pathological review may be required for correct diagnoses. This is necessary for appropriate management and to establish an association with hereditary cancer syndromes. The work was supported by the Ministry of Health of the Czech Republic - Conceptual Development of Research Organization, Faculty Hospital of Ostrava /2015. We thank to Lenka Foretová, M.D., Ph.D., (MMCI, Brno) and Radoslava Tomanová, M.D., (Institute of Pathology, University Hospital Ostrava) for rewarding advice, Mrs. Jana Němcová (Department of Medical Genetics, University Hospital Ostrava), Bc. Ludmila Stuchlá and Mrs. Lenka Zivčáková (Medical Library, University Hospital Ostrava) for help during manuscript preparation. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 10. 3. 2019 Accepted: 16. 4. 2019.

An Update on Inherited Colon Cancer and Gastrointestinal Polyposis.

BACKGROUND: It is estimated that 5-10% of colorectal cancers arise due to a known genetic syndrome. Individuals with these cancer syndromes are also at risk of extracolonic cancers. Polyposis and nonpolyposis hereditary syndromes are generally recognized. Inclusion of next-generation sequencing technology, especially multiple-gene panel testing, in routine laboratory practice has made identifying the causes of these diseases significantly easier. PURPOSE: To summarize current knowledge of the causes, clinical manifestations, diagnostic criteria, and recommendations for presymptomatic screening of individuals at risk of hereditary gastrointestinal polyposis and colorectal cancer syndromes. We dicuss currently defined syndromes detected by multiple-gene panel next-generation sequencing; these include constitutional mismatch repair deficiency (biallelic MLH1, MSH2, MSH6, PMS2 gene mutations), gastric adenocarcinoma and proximal polyposis of the stomach (APC gene), NTHL1-associated polyposis, polymerase proofreading-associated polyposis (POLD1, POLE genes), juvenile polyposis (SMAD4, BMPR1A genes), and serrated polyposis syndromes. Another aim is to summarize recent knowledge about well-known syndromes, including hereditary nonpolyposis colon cancer (Lynch syndrome), familial adenomatous polyposis, MUTYH-associated polyposis, and Peutz-Jeghers and Cowden/PTEN hamartoma tumor syndromes. CONCLUSION: Awareness of hereditary polyposis/colon cancer syndromes enables early diagnosis and prevention of cancer in affected individuals and their relatives. Genetic counseling, presymptomatic testing of at-risk individuals, and efficient screening may be beneficial for affected families. Thank to Lenka Foretová, M.D., PhD, (Masaryk Memorial Cancer Institute, Brno) for a critical review of the manuscript and valuable advices. The author declares she has no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 6. 6. 2019.

Genetic Aetiology of Nonsyndromic Hearing Loss in Moravia-Silesia.

BACKGROUND AND OBJECTIVE: Hearing loss is the most common sensory deficit in humans. The aim of this study was to clarify the genetic aetiology of nonsyndromic hearing loss in the Moravian-Silesian population of the Czech Republic. PATIENTS AND METHODS: This study included 200 patients (93 males, 107 females, mean age 16.9 years, ranging from 4 months to 62 years) with nonsyndromic sensorineural hearing loss. We screened all patients for mutations in GJB2 and the large deletion del(GJB6-D13S1830). We performed further screening for additional genes (SERPINB6, TMIE, COCH, ESPN, ACTG1, KCNQ4, and GJB3) with Sanger sequencing on a subset of patients that were negative for GJB2 mutations. RESULTS: We detected biallelic GJB2 mutations in 44 patients (22%). Among these patients, 63.6%, 9.1% and 2.3% exhibited homozygous c.35delG, p.Trp24*, and p.Met34Thr mutations, respectively. The remaining 25% of these patients exhibited compound heterozygous c.35delG, c.-23+1G>A, p.Trp24*, p.Val37Ile, p.Met34Thr, p.Leu90Pro, c.235delC, c.313_326del14, p.Ser139Asn, and p.Gly147Leu mutations. We found a monoallelic GJB2 mutation in 12 patients (6.6%). We found no pathogenic mutations in the other tested genes. Conclusions: One fifth of our cohort had deafness related to GJB2 mutations. The del(GJB6-D13S1830), SERPINB6, TMIE, COCH, ESPN, ACTG1, GJB3, and KCNQ4 mutations were infrequently associated with deafness in the Moravian-Silesian population. Therefore, we suggest that del(GJB6-D13S1830) testing should be performed only when patients with deafness carry the monoallelic GJB2 mutation.

STRC Deletion is a Frequent Cause of Slight to Moderate Congenital Hearing Impairment in the Czech Republic.

OBJECTIVE: This study aimed to clarify the molecular epidemiology of hearing loss by identifying the responsible genes in patients without GJB2 mutations. STUDY DESIGN: Prospective genetic study. SETTING: Tertiary referral hospital. PATIENTS: Fifty one patients with bilateral sensorineural hearing loss, 20 men, and 31 women, mean age 24.9 years, range 3 to 64 years, from 49 families. GJB2 and deltaGJB6-D13S1830 mutations were excluded previously. INTERVENTION: Diagnostic. Sixty-nine genes reported to be causative of hearing loss were analyzed. Sequence capture technology, next-generation sequencing, and multiplex ligation-dependent probe amplification (MLPA) were used. Coverage of STRC was screened in Integrative Genomics Viewer software. MAIN OUTCOME MEASURE: Identification of causal pathogenic mutations in genes related to deafness. RESULTS: Five families (10%) had recessive STRC deletions or mutations. Five unrelated patients (10%) had recessive mutations in TMPRSS3, USH2A, PCDH15, LOXHD1, and MYO15A. Three families (6%) had autosomal dominant mutations in MYO6A, KCNQ4, and SIX1. One family (2%) had an X-linked POU3F4 mutation. Thus, we identified the cause of hearing loss in 28% of the families studied. CONCLUSIONS: Following GJB2, STRC was the second most frequently mutated gene in patients from the Czech Republic with hearing loss. To decrease the cost of testing, we recommend STRC deletion screening with MLPA before next-generation sequencing. The existence of a pseudogene and polymorphic STRC regions can lead to false-positive or false-negative results when copy number variation analysis is based on next-generation sequencing data.

Familial hematuria: A review.

The most frequent cause of familial glomerular hematuria is thin basement membrane nephropathy (TBMN) caused by germline COL4A3 or COL4A4 gene mutations. Less frequent but important cause with respect to morbidity is Alport syndrome caused by germline COL4A5 gene mutations. The features of Alport syndrome include hematuria, proteinuria and all males with X-linked disease and all individuals with recessive disease will develop end stage renal disease, usually at early youth. In X-linked Alport syndrome, a clear genotype-phenotype correlation is typically observed in men. Deleterious COL4A5 mutations are associated with a more severe renal phenotype and more frequent high-frequency sensorineural hearing loss and ocular abnormalities. Less severe COL4A5 mutations result in a milder phenotype, with less frequent and later onset extrarenal anomalies. The phenotype in females is highly variable, mostly due to inactivation of one of the X chromosomes. Isolated cases may be caused by de novo COL4A5 mutations or by gonosomal mosaicism. Untreated autosomal recessive Alport syndrome, caused by COL4A3 and COL4A4 mutations, is typically associated with ESRD at the age of 23-25 years and extrarenal symptoms in both men and women. The TBMN phenotype is associated with heterozygous carriers of COL4A3, COL4A4 mutations. Molecular genetic testing is the gold standard for diagnosing these diseases. Although genotype-phenotype correlations exist, the phenotype is influenced by modifying factors, which remain mainly undefined. No therapy is available that targets the cause of Alport syndrome; angiotensin-converting enzyme inhibitor therapy delays renal failure and improves lifespan.

Increased levels of MMP-3, MMP-9 and MPO represent predictors of in-stent restenosis, while increased levels of ADMA, LCAT, ApoE and ApoD predict bare metal stent patency.

AIMS: We sought to identify biochemical predictors that indicate susceptibility to in-stent restenosis (ISR) after coronary artery bare-metal stenting. METHODS: A total of 111 consecutive patients with post-percutaneous coronary intervention (PCI) in-stent restenosis of a target lesion within 12 months were matched for age, sex, vessel diameter, and diabetes with 111 controls without post-PCI ISR. Plasma or serum levels of biochemical markers were measured: matrix metalloproteinases (MMP) 2, 3, 9; myeloperoxidase (MPO); asymmetric dimethylarginine (ADMA); lipoprotein (a) (Lp[a]); apolipoproteins E and D (ApoE and D); and lecitin-cholesterol acyltransferase (LCAT). Multivariable logistic regression association tests were performed. RESULTS: Increased plasma MMP-3 (OR: 1.013; 95% CI: 1.004-1.023; P = 0.005), MMP-9 (OR: 1.014; 95% CI: 1.008-1.020; P < 0.0001) or MPO (OR: 1,003; 95% CI: 1.001-1.005; P = 0.002) was significantly associated with increased risk of ISR. Increased levels of ADMA (OR: 0.212; 95% CI: 0.054-0.827; P = 0.026), ApoE (OR: 0.924; 95% CI: 0.899-0.951; P < 0.0001), ApoD (OR: 0.919; 95% CI: 0.880-0.959; P = 0.0001), or LCAT (OR: 0.927; 95% CI: 0.902-0.952; P < 0.0001) was associated with risk reduction. No correlation was found between plasma MMP-2 or Lp (a) and ISR risk. CONCLUSIONS: Increased levels of MMP-3, MMP-9, and MPO represent predictors of ISR after bare-metal stent implantation. In contrast, increased ADMA, LCAT, and Apo E and D indicate a decreased in-stent restenosis occurrence.

Reference intervals of plasma matrix metalloproteinases 2, 3, and 9 and serum asymmetric dimethylarginine levels.

OBJECTIVE: The present study aimed to verify the reference intervals of plasma matrix metalloproteinases (MMPs) 2, 3, and 9 and serum asymmetric dimethylarginine (ADMA) in a healthy population with an average age corresponding to that of patients with cardiovascular diseases. METHODS: The study included 180 healthy volunteers. Plasma MMP-2, MMP-3, MMP-9, and serum ADMA levels were determined using an enzyme-linked immunosorbent assay. These levels were analyzed for association with age and gender. The Cbstat5, R software, and NCSS 2007 programs were used for statistical analysis. RESULTS: The average volunteer age was 47.4 years in the group in which MMP-3 and ADMA were analyzed, 40.3 years in the MMP-9 group, and 47.8 years for the MMP-2 group. Serum ADMA levels were determined to be independent of age and gender. Plasma MMP-2 levels were significantly correlated with age (p = 0.001), with lower levels detected in persons ≤ 49 years of age. Plasma MMP-3 was significantly associated with both age (p < 0.0001) and gender, with lower levels detected in persons of ≤ 47 years of age and among women. Plasma MMP-9 levels were not age dependent, but were associated with gender (p = 0.014), showing lower levels in women. CONCLUSIONS: Reference intervals of heparin-plasma MMP-2, MMP-3, and MMP-9 and serum ADMA levels were determined. MMP-2 and MMP-3 levels were found to be age dependent, and MMP-3 and MMP-9 levels were gender dependent.

HCFC1 loss-of-function mutations disrupt neuronal and neural progenitor cells of the developing brain.

Both gain- and loss-of-function mutations have recently implicated HCFC1 in neurodevelopmental disorders. Here, we extend our previous HCFC1 over-expression studies by employing short hairpin RNA to reduce the expression of Hcfc1 in embryonic neural cells. We show that in contrast to over-expression, loss of Hcfc1 favoured proliferation of neural progenitor cells at the expense of differentiation and promoted axonal growth of post-mitotic neurons. To further support the involvement of HCFC1 in neurological disorders, we report two novel HCFC1 missense variants found in individuals with intellectual disability (ID). One of these variants, together with three previously reported HCFC1 missense variants of unknown pathogenicity, were functionally assessed using multiple cell-based assays. We show that three out of the four variants tested result in a partial loss of HCFC1 function. While over-expression of the wild-type HCFC1 caused reduction in HEK293T cell proliferation and axonal growth of neurons, these effects were alleviated upon over-expression of three of the four HCFC1 variants tested. One of these partial loss-of-function variants disrupted a nuclear localization sequence and the resulting protein displayed reduced ability to localize to the cell nucleus. The other two variants displayed negative effects on the expression of the HCFC1 target gene MMACHC, which is responsible for the metabolism of cobalamin, suggesting that these individuals may also be susceptible to cobalamin deficiency. Together, our work identifies plausible cellular consequences of missense HCFC1 variants and identifies likely and relevant disease mechanisms that converge on embryonic stages of brain development.

[Familiar Mediterranean fever in Czech Republic]. / Familiární stredozemní horecka v Ceské republice.

Familiar Mediterranean fever (FMF) is a well defined autosomal recessive disease occurring mostly in Mediterranean regions. Here we present the experience from one center from Czech Republic, where we follow 4 families with patients with genetically proven FMF. Three out of these 4 families cluster to one limited region in Moravia, in the heart of Europe, without any linkage to Mediterranean origin. Furthermore, majority of these patients are heterozygots presenting with well defined typical clinical symptoms. Potential pseudodominant inheritance and/or epigenetic and environmental factors might influence clinical presentation of the disease.

Spinocerebellar ataxias type 8, 12, and 17 and dentatorubro-pallidoluysian atrophy in Czech ataxic patients.

Spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders currently associated with 27 genes. The most frequent types are caused by expansions in coding CAG repeats. The frequency of SCA subtypes varies among populations. We examined the occurrence of rare SCAs, SCA8, SCA12, SCA17 and dentatorubro-pallidoluysian atrophy (DRPLA), in the Czech population from where the data were missing. We analyzed causal gene expansions in 515 familial and sporadic ataxic patients negatively tested for SCA1-3 and SCA6-7. Pathogenic SCA8 and SCA17 expansions were identified in eight and five patients, respectively. Tay-Sachs disease was later diagnosed in one patient with an SCA8 expansion and the diagnosis of multiple sclerosis (MS) was suspected in two other patients with SCA8 expansions. These findings are probably coincidental, although the participation of SCA8 expansions in the susceptibility to MS and disease progression cannot be fully excluded. None of the patients had pathogenic SCA12 or DRPLA expansions. However, three patients had intermediate SCA12 alleles out of the normal range with 36 and 43 CAGs. Amyotrophic lateral sclerosis (ALS) was probable in the patient with 43 CAGs. This coincidence is remarkable, especially in the context with the recently identified predisposing role of longer SCA2 alleles in ALS. Five families with SCA17 represent a significant portion of ataxic patients and this should be reflected in the diagnostics of SCAs in the Czech population. SCA8 expansions must be considered after careful clinical evaluation.

Four novel point mutations in the PMP22 gene with phenotypes of HNPP and Dejerine-Sottas neuropathy.

We report four novel point mutations in the PMP22 gene with two different phenotypes: mutation p.Ser79Thr arose de novo in a patient with the Dejerine-Sottas neuropathy (DSN) phenotype; and mutations c.78+5 G>A, c.320-1 G>C, and p.Trp140Stop segregated with HNPP in 5 families.Our findings show that point mutations in PMP22 may be more likely in HNPP patients than in CMT1 patients after exclusion of CMT1A/HNPP.

Elejalde syndrome--a case report.

Elejalde syndrome (McKusick 200995), also known as acrocephalopolydactylous dysplasia, is a rare condition. We describe a sixth patient with this syndrome which is characterized by craniosynostosis and hyperproliferation of fibroblasts in many tissues including skin, liver, kidney, and pancreas. The cause of the syndrome is the homozygous state of an autosomal recessive mutation. We present a hypothesis that Elejalde syndrome might be associated with an inactivating FGFR gene mutation.

Prognostic value of hMLH1 and hMSH2 immunohistochemical expression in non-small cell lung cancer. A tissue microarray study.

BACKGROUND: hMLH1 and hMSH2 genes are both known to play a role in DNA mismatch repair. Nonetheless, the clinical significance of hMLH1 and hMSH2 protein expression in lung cancers remains unclear. AIM: The aim of this study was to investigate the immunohistochemical expression of hMLH1 and hMSH2 proteins in tumor specimens from 179 non-small cell lung cancer (NSCLC) patients using a tissue microarray technique and to correlate these results with other clinicopathological variables, including the disease specific and overall survivals. METHOD: hMLH1 and hMSH2 protein expression was evaluated by immunohistochemistry using monoclonal antibodies G168-728 for hMLH1 and FE11 for hMSH2 protein expression analysis. The Pearson chi2 test was used to compare the hMLH1 and hMSH2 alterations among the cases and between various clinical and laboratory variables. P < or = 0.05 was considered statistically significant. RESULTS: Alteration of hMLH1 and hMSH2 protein expression was observed in 10 % of patients. No significant correlation was found between the protein expression and patient age, smoking status, tumor histology or disease stage and disease free and overall survival. CONCLUSIONS: Alterations in the expression of hMLH1 and hMSH2 proteins did not have any prognostic value in stage III. NSCLC patients.

Identification of five new families strengthens the link between childhood choroid plexus carcinoma and germline TP53 mutations.

We present five families of paediatric patients suffering from choroid plexus carcinoma in which we found germline TP53 mutations. Only one of the families conformed to the criteria of Li-Fraumeni syndrome and only three (including the Li-Fraumeni syndrome family) met the Chompret criteria for germline TP53 mutation testing. In the remaining two families no family history of cancer was identified and/or the parents of the patient were shown not to carry the mutation. Our results give further support to the notion that the occurrence of this rare paediatric tumour, especially in combination with a positive family history of cancer, but possibly also without any family history, may be an indicator of a germline TP53 mutation. The identification of this genetic defect has important consequences for cancer prevention and treatment in affected families.

Expression of the hMLH1 and hMSH2 proteins in normal tissues: relationship to cancer predisposition in hereditary non-polyposis colon cancer.

The majority of tumours in patients with hereditary non-polyposis colon cancer (HNPCC) occur in large intestine and endometrium; also, other tissues are at increased risk. We studied expression of hMLH1 and hMSH2 proteins in 148 normal samples of various tissues from non-HNPCC patients and in 14 normal colon tissues from HNPCC patients. Immunohistochemical technique was used. Intensity of nuclear staining, percentage of stained cells and H-scores were calculated. Tissues were divided into groups. Groups A, B and C included tissues with increased risk of cancer in HNPCC A) stomach, small and large bowel; (B) endometrium; (C) ovary, ureter, urinary bladder, kidney and liver. Group D tissues were without increased risk. Expression of the proteins was significantly higher in groups A, B and C compared with group D (P<0.0001, P=0.0004 for hMSH2 in C versus D). The expression was highest in testis. In colons of HNPCC patients, expression of the mutated gene product was significantly lower than in non-HNPCC patients. In conclusion, hMLH1/hMSH2 protein expression is constitutively higher in certain cell types of certain tissues, including the majority of tissues that are at increased risk of cancer in HNPCC. However, association of strong hMLH1/hMSH2 expression with cancer risk is not strictly valid.