Modelling Germline Mosaicism and Different New Mutation Rates Simultaneously for Appropriate Risk Calculations in Families with Duchenne Muscular Dystrophy

For several genetic diseases two biological phenomena have been recognised as important: germline mosaicism; and different new mutation rates in males and females depending on mutation type. Both principles have been investigated separately and their influence on risk estimation in families has been exemplified in the literature. The aim of this paper is to present a general model that includes mosaicism and different new mutation rates. Mosaicism is introduced by defining additional alleles at the disease locus in combination with adapted segregation rules. Taking Duchenne muscular dystrophy as an example, we derive the conditions which have to be fulfilled for a population in mutation selection equilibrium. Our approach describes the model at the population level and not in individual subjects. This has the advantage of being able to use well known algorithms for the calculation of likelihoods in pedigrees, and to include additional diagnostic information such as marker genotypes and carrier deletion test results. We demonstrate the impact of the new model on a typical pedigree. In families where the patient is not available, the distinction between point mutations and deletions is important, since often molecular diagnostic tests for females can only screen for deletions. Negative deletion test results can now be included in the risk calculations.     

Risk estimation as a decision-making tool for genetic analysis of the breast cancer susceptibility genes. EC Demonstration Project on Familial Breast Cancer

For genetic counselling of a woman on familial breast cancer, an accurate evaluation of the probability that she carries a germ-line mutation is needed to assist in making decisions about genetic-testing. We used data from eight collaborating centres comprising 618 families (346 breast cancer only, 239 breast or ovarian cancer) recruited as research families or counselled for familial breast cancer, representing a broad range of family structures. Screening was performed in affected women from 618 families for germ-line mutations in BRCA1 and in 176 families for BRCA2 mutations, using different methods including SSCP, CSGE, DGGE, FAMA and PTT analysis followed by direct sequencing. Germ-line BRCA1 mutations were detected in 132 families and BRCA2 mutations in 16 families. The probability of being a carrier of a dominant breast cancer gene was calculated for the screened individual under the established genetic model for breast cancer susceptibility, first, with parameters for age-specific penetrances for breast cancer only [7] and, second, with age-specific penetrances for ovarian cancer in addition [20]. Our results indicate that the estimated probability of carrying a dominant breast cancer gene gives a direct measure of the likelihood of detecting mutations in BRCA1 and BRCA2. For breast/ovarian cancer families, the genetic model according to Narod et al. [20] is preferable for calculating the proband's genetic risk, and gives detection rates that indicate a 50% sensitivity of the gene test. Due to the incomplete BRCA2 screening of the families, we cannot yet draw any conclusions with respect to the breast cancer only families.     

'Indirect' BRCA1/2 testing: a useful approach in hereditary breast and ovarian cancer families without a living affected relative

We report an approach for BRCA1/2 testing whereby genetic testing can be offered to families at high risk of hereditary breast and ovarian cancer but where no DNA from affected relatives is available. By testing two or more unaffected relatives at 50% risk of being heterozygous for a potential BRCA1/2 mutation, there is a chance of up to 99% of finding a mutation that would have been detectable in an affected individual from the same family. The overall likelihood of identifying a mutation is dependent on the family history, and therefore 'indirect' testing would be most applicable for families with a very high risk of carrying a BRCA1/2 mutation. Using this approach also requires balancing issues of testing resource limitations, family dynamics and adequate preparation of unaffected persons for a positive test, with the advantages of targeting screening and prophylactic surgery.     

Complex segregation analysis and computer-assisted genetic risk assessment for Duchenne muscular dystrophy

Two hundred forty-four Toronto pedigrees of Duchenne muscular dystrophy patients have been partitioned into nuclear families with pointers for complex segregation analysis under a mixed model. The model takes into account the major X-linked locus and a multifactorial transmissible component for creatine kinase activity in females. The incidence in the province of Ontario is estimated to be 292 per million male births. The proportion of sporadic cases is 1/3, demonstrating equal mutation rates in males and females. A multifactorial component (H = 0.379) contributes to family resemblance for creatine kinase measurements. Examples are presented of the application of a computer program, COUNSEL, to derive genetic risks for genetic counseling with consideration of the multifactorial component.     

A genetic model for determining MSH2 and MLH1 carrier probabilities based on family history and tumor microsatellite instability

Mutation-predicting models can be useful when deciding on the genetic testing of individuals at risk and in determining the cost effectiveness of screening strategies at the population level. The aim of this study was to evaluate the performance of a newly developed genetic model that incorporates tumor microsatellite instability (MSI) information, called the AIFEG model, and in predicting the presence of mutations in MSH2 and MLH1 in probands with suspected hereditary non-polyposis colorectal cancer. The AIFEG model is based on published estimates of mutation frequencies and cancer penetrances in carriers and non-carriers and employs the program MLINK of the FASTLINK package to calculate the proband's carrier probability. Model performance is evaluated in a series of 219 families screened for mutations in both MSH2 and MLH1, in which 68 disease-causing mutations were identified. Predictions are first obtained using family history only and then converted into posterior probabilities using information on MSI. This improves predictions substantially. Using a probability threshold of 10% for mutation analysis, the AIFEG model applied to our series has 100% sensitivity and 71% specificity.     

Autosomal dominant nocturnal frontal lobe epilepsy: a genotypic comparative study of Japanese and Korean families carrying the CHRNA4 Ser284Leu mutation

Autosomal dominant nocturnal frontal lobe epilepsy is a familial partial epilepsy syndrome and the first human idiopathic epilepsy known to be related to specific gene defects. Clinically available molecular genetic testing reveals mutations in three genes, CHRNA4, CHRNB2 and CHRNA2. Mutations in CHRNA4 have been found in families from different countries; the Ser280Phe in an Australian, Spanish, Norwegian and Scottish families, and the Ser284Leu in a Japanese, Korean, Polish and Lebanese families. Clear evidence for founder effect was not reported among them, including a haplotype study carried out on the Australian and Norwegian families. Japanese and Koreans, because of their geographical closeness and historical interactions, show greater genetic similarities than do the populations of other countries where the mutation is found. Haplotype analysis in the two previously reported families showed, however, independent occurrence of the Ser284Leu mutation. The affected nucleotide was highly conserved and associated with a CpG hypermutable site, while other CHRNA4 mutations were not in mutation hot spots. Association with a CpG site accounts for independent occurrence of the Ser284Leu mutation.     

Mutation and linkage disequilibrium analysis in genetic counselling of Spanish cystic fibrosis families.

We have analysed haplotypes for four DNA polymorphisms, closely linked to the cystic fibrosis (CF) gene, in 82 Spanish families, in which the CF probands are either homozygous for non-delta F508 mutations or heterozygous for the delta F508 deletion and other CF mutations. The analysis provides genetic data for a new polymorphism for the closely linked marker pKM.19, which is very strongly associated with CF. Haplotypes generated with the four marker loci are also in strong disequilibrium with the non-delta F508 CF chromosomes. The data reported here are useful in 1 in 4 risk pregnancies of parents who have no living affected child, and when counselling close relatives of CF families who are negative for the major CF mutation. The data presented are useful in our population, in which the majority of CF mutations, apart from the delta F508 deletion, are uncommon. For other populations in which mutation heterogeneity is also very high, it still might be more feasible to use RFLPs for diagnostic purposes, when analysis for common mutations is negative and DNA is available from the index patient. The experience presented here provides a model for these population groups who in turn should obtain their own haplotype data. In addition, the model system for genetic counselling presented here might also be useful for other genetic disorders.     

Arg120stop nonsense mutation in the RP2 gene: mutational hotspot and germ line mosaicism?

Mutations in the RP2 gene account for up to 20% of X-linked recessive retinitis pigmentosa (RP). Arg120stop is to date the most frequently reported mutation found in RP2. Mutation screening was performed during the course of a large screening program of retinal degenerative disorders (RDDs) in South Africa using exon 1 and 2 of RP2 in 20 unrelated families with an X-linked mode of retinal degenerative inheritance. Direct sequencing analysis revealed a C-->T transition at position 358 in the proband in a family of German origin. Subsequent analysis revealed that this Arg120stop mutation cosegregated with the disease in an additional affected family member. The nonsense mutation, Arg120stop, could not however, be detected in the somatic cells of the obligate carrier female. This, the first report of a germ line mutation for a family with RP, has many implications for genetic counseling of retinal degeneration (RD). To avoid inaccurate risk assessment for RP due to epigenetic events, such as the rare occurrence of germ line mosaicism, genetic counseling in families with XLRP should always be guided by molecular testing.     

New options for prenatal diagnosis in autosomal recessive polycystic kidney disease by mutation analysis of the PKHD1 gene

Due to the poor prognosis of severe autosomal recessive polycystic kidney disease (ARPKD), there is a strong demand for prenatal diagnosis (PD). Reliable PD testing is possible by molecular genetic analysis only. Although haplotype-based analysis is feasible in most cases, it is associated with a risk of misdiagnosis in families without pathoanatomically proven diagnosis. Linkage analysis is impossible in families where DNA of the index patient is not available. Direct mutation analysis of the recently identified polycystic kidney and hepatic disease 1 gene opens new options in families to whom a reliable PD cannot be offered on the basis of linkage analysis. We for the first time report two cases with PD based on mutation detection, illustrating the new options for PD in ARPKD.     

Analysis of CGG variation through 642 meioses in Fragile X families

Fragile X syndrome is the commonest familial form of inherited mental retardation. The molecular defect is an expansion of the CGG trinucleotide repeats in the 5' untranslated region of the FMR1 gene that is inherited in an unstable fashion in fragile X families. In an attempt to provide more information about the CGG tract intergenerational variation, we have evaluated 642 transmissions in 175 Fragile X families. PCR and Southern blot (StB12.3) was used to analyse the CGG number. Among premutated alleles, 90.2% showed expansion, two-thirds to a full mutation while the rest remained in the premutation range, 5.5% of alleles did not vary and finally 4.3% of them reduced in size. Premutated females showed an increased risk of expansion to the full mutation depending on the CGG tract. The estimated risk for 80 triplets is more than seven times that of a woman carrying 59 CGG, the risk being 100% for alleles of >100 repeats. Fifty-nine repeats was the smallest allele that expanded to full mutation. Contractions were detected more frequently in males than in females, being statistically significant. This study contributes to the literature by increasing the data available regarding transmissions in Fragile X families and it allows us to perform more precise genetic counselling for women with the CGG repeat in the premutation range.     

Genetic screening for autosomal recessive nonsyndromic mental retardation in an isolated population in Israel

Nonsyndromic mental retardation (NSMR) is the diagnosis of exclusion in mentally retarded individuals without additional abnormalities. We have recently identified a protein-truncating mutation, G408fsX437, in the gene CC2D1A on chromosome 19p13.12 in nine consanguineous Israeli Arab families with severe autosomal recessive NSMR, and have developed a comprehensive prevention program among the at-risk population in the village. The subjects tested were healthy women who were invited to undergo the genetic screening test as a part of their routine pregnancy monitoring. One hundred and seventeen subjects reported a family history positive for mental retardation. We tested 524 pregnant or preconceptional women and found 47 carriers (approximately 1/11), whose spouses were then recommended to undergo testing. We identified eight carrier couples, who were given genetic counseling and offered prenatal diagnosis. Of all the marriages, 28.6% were consanguineous; 16.5% of the total were between first cousins. The high prevalence of the mutation can be explained both by the founder effect owing to the generally high consanguinity rate among the inhabitants of the village, and also because two families with excessive numbers of mentally retarded offspring were unacceptable as marriage partners by the rest of the families. This is the first example of the establishment of a large-scale genetic screening program for autosomal recessive NSMR, which was made possible owing to the high frequency of the specific causative mutation in this isolated population.     

Costs and benefits of diagnosing familial breast cancer

Based on results from our surveillance program for women at risk for inherited breast cancer, we have calculated cost per year earned. Norwegian National Insurance Service reimbursement fees were used in the calculations. The calculated costs are based on empirical figures for expanding already established medical genetic departments and diagnostic outpatient clinics to undertake the work described. Cost per year earned was estimated at Euro 753 using our current practice of identifying the high-risk women through a traditional cancer family clinic. A strategy of identifying the high-risk families through genetic testing of all incident breast and ovarian cancers for founder mutations in BRCA1, will increase the cost to Euro 832. Costs related more to genetic counseling and clinical follow-up than to laboratory procedures. This potential economic limiting factor coincides with a shortage of personnel trained in genetic counseling. The number of relatives counseled to identify one healthy female mutation carrier (i.e. the uptake of genetic testing) is more important to cost-effectiveness than family size. Costs will vary depending upon the penetrance of the mutations detected and the prevalence of founder mutations in the population examined. Prevalences of BRCA1 founder mutations in some high incidence areas of Norway may be sufficiently high to consider population screening. Unlike mutation screening of cancer genes, founder mutation analysis will not identify DNA variants of uncertain clinical significance. Identification of high-risk families through founder mutation analysis of BRCA1 ensures that families with maximum risks are given first access to the limited resources of the high-risk clinics. This may be the greatest contribution to increased cost effectiveness of such a strategy. The assumptions underlying the calculations are discussed. The conclusion is that inherited breast cancer may be managed effectively for the cost of Euro 750-1,600 per year earned.     

New York State screening program for fragile X syndrome: a progress report.

New York State has established a program to screen post-pubertal mentally retarded males for the fragile X [fra(X)] syndrome. The goal of the program is to identify affected males and inform their families of the diagnosis. Females in these families who are at risk for inheriting the mutation will then be able to determine their carrier status and consider that information in making reproductive decisions. Males were evaluated for 10 features of the syndrome by physicians and nurses throughout the state; cytogenetic analysis was carried out on a subset of this population. A total of 1332 males has been screened and chromosome studies have been completed for 489. Forty-three (9%) were positive for fra(X), and an additional 11 other chromosome abnormalities were identified. The 43 patients belonged to 38 families. Of the 24 families who were informed of the diagnosis, 12 consulted genetic counseling centers for follow-up studies and 12 did not.     

DUCHEN: an interactive computer program for calculating heterozygosity (carrier) risks in X-linked recessive lethal diseases, and its application in Duchenne muscular dystrophy

The program DUCHEN calculates the probability that a woman is a carrier of an X-linked, lethal recessive disease on the basis of information in the woman's family and any available biochemical data. It is easily used by persons without computer knowledge or experience. The present version can accommodate families consisting of up to 100 people in seven generations. Risks may be estimated on the basis of pedigree information only, or with the inclusion of one or more types of biochemical test results. Biochemical data are incorporated with pedigree information into final risks using the powerful statistical technique of logistic discrimination, a procedure particularly suited for the separation of non-normal populations on the basis of overlapping quantitative characteristics. Mutation rates are specified separately for males and females. DUCHEN is available in FORTRAN 77, IBM BASIC, and Applesoft BASIC, and may be used on a variety of mainframe or microcomputers. The model was used to calculate risks for 375 girls and women in 46 families with Duchenne muscular dystrophy (DMD); serum creatine kinase tests had been carried out on 167 of these subjects who were of reproductive age. Carrier probabilities equal to or lower than the population risk (0.0004) were obtained for 21% of the aunts and 43% of the cousins of affected boys from families with an isolated case of DMD and for 14% of the cousins of affected boys from families with a known DMD history. DUCHEN should assist counsellors in determining which members of large families should be further examined using either standard biochemical carrier detection methods or DNA marker studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cancer risks in BRCA2 families: estimates for sites other than breast and ovary

Background: In BRCA2 mutation carriers, increased risks have been reported for several cancer sites besides breast and ovary. As most of the families included in earlier reports were selected on the basis of multiple breast/ovarian cancer cases, it is possible that risk estimates may differ in mutation carriers with a less striking family history. Methods: In the Netherlands, 139 BRCA2 families with 66 different pathogenic mutations were included in a nationwide study. To avoid testing bias, we chose not to estimate risk in typed carriers, but rather in male and female family members with a 50% prior probability of being a carrier (n = 1811). The relative risk (RR) for each cancer site with the exception of breast and ovarian cancer was determined by comparing observed numbers with those expected, based on Dutch cancer incidence rates. Results: We observed an excess risk for four cancer sites: pancreas (RR 5.9; 95% confidence interval (CI) 3.2 to 10.0), prostate (2.5; 1.6 to 3.8), bone (14.4; 2.9 to 42.1) and pharynx (7.3; 2.0 to 18.6). A small increase was observed for cancer of the digestive tract (1.5; 1.1 to 1.9). Histological verification was available for 46% of the tumours. Nearly all increased risks reached statistical significance for men only. Cancer risks tended to be higher for people before the age of 65 years. Moreover, families with mutations outside the previously defined ovarian cancer cluster region tended to have a higher cancer risk. Conclusions: We found that BRCA2 carriers are at increased risk for cancers of the prostate and pancreas, and possibly bone and pharynx. Larger databases with extended follow up are needed to provide insight into mutation specific risks of selected carriers in BRCA2 families.     

Screening for the BRCA1-ins6kbEx13 mutation: potential for misdiagnosis. Mutation in brief #964. Online

Misdiagnosis of a germline mutation associated with an inherited disease syndrome can have serious implications for the clinical management of patients. A false negative diagnosis (mutation missed by genetic screening) limits decision making about intervention strategies within families. More serious is the consequence of a false positive diagnosis (genetic test suggesting a mutation is present when it is not). This could lead to an individual, falsely diagnosed as a mutation carrier, undergoing unnecessary clinical intervention, possibly involving risk-reducing surgery. As part of screening 283 ovarian cancer families for BRCA1 mutations, we used two different methods (mutation specific PCR and multiplex ligation-dependent probe amplification) to screen for a known rearrangement mutation L78833.1:g.44369_50449dup (ins6kbEx13). We found false positive and false negative results in several families. We then tested 61 known carriers or non-carriers from an epidemiological study of BRCA1 and BRCA2 mutation carriers (the EMBRACE study). These data highlight the need for caution when interpreting analyses of the ins6kbEx13 mutation and similar mutations, where characterising the exact sequence alteration for a deleterious mutation is not a part of the routine genetic test.     

Mitochondrial disorders: genetics, counseling, prenatal diagnosis and reproductive options

Most patients with mitochondrial disorders are diagnosed by finding a respiratory chain enzyme defect or a mutation in the mitochondrial DNA (mtDNA). The provision of accurate genetic counseling and reproductive options to these families is complicated by the unique genetic features of mtDNA that distinguish it from Mendelian genetics. These include maternal inheritance, heteroplasmy, the threshold effect, the mitochondrial bottleneck, tissue variation, and selection. Although we still have much to learn about mtDNA genetics, it is now possible to provide useful guidance to families with an mtDNA mutation or a respiratory chain enzyme defect. We describe a range of current reproductive options that may be considered for prevention of transmission of mtDNA mutations, including the use of donor oocytes, prenatal diagnosis (by chorionic villus sampling or amniocentesis), and preimplantation genetic diagnosis, plus possible future options such as nuclear transfer and cytoplasmic transfer. For common mtDNA mutations associated with mitochondrial cytopathies (such as NARP, Leigh Disease, MELAS, MERRF, Leber's Hereditary Optic Neuropathy, CPEO, Kearns-Sayre syndrome, and Pearson syndrome), we summarize the available data on recurrence risk and discuss the relative advantages and disadvantages of reproductive options.     

Problems in genetic counseling in a family with an “atypical” centronuclear myopathy

Genetic counseling often deals with a rare disease the inheritance of which is not clearly established or in which genetic heterogeneity is reported. In addition, relevant parameters such as penetrance, gene frequency, and mutation rate may not be available. In this situation, establishing the risk may be very difficult. An example is presented in which Bayesian risk calculation proved to be of great help in providing precise risk estimates in a family in which an “atypical” centronuclear myopathy was segregating.     

Problems in genetic counseling in a family with an "atypical" centronuclear myopathy

Genetic counseling often deals with a rare disease the inheritance of which is not clearly established or in which genetic heterogeneity is reported. In addition, relevant parameters such as penetrance, gene frequency, and mutation rate may not be available. In this situation, establishing the risk may be very difficult. An example is presented in which Bayesian risk calculation proved to be of great help in providing precise risk estimates in a family in which an "atypical" centronuclear myopathy was segregating.