The hemoglobinopathies, molecular disease mechanisms and diagnostics.

Hemoglobinopathies are the most common monogenic disorders in the world with an ever increasing global disease burden each year. As most hemoglobinopathies show recessive inheritance carriers are usually clinically silent. Programmes for preconception and antenatal carrier screening, with the option of prenatal diagnosis are considered beneficial in many endemic countries. With the development of genetic tools such as Array analysis and Next Generation Sequencing in addition to state of the art screening at the hematologic, biochemic and genetic level, have contributed to the discovery of an increasing number of rare rearrangements and novel factors influencing the disease severity over the recent years. This review summarizes the basic requirements for adequate carrier screening analysis, the importance of genotype-phenotype correlation and how this may lead to the unrevealing exceptional interactions causing a clinically more severe phenotype in otherwise asymptomatic carriers. A special group of patients are ß-thalassemia carriers presenting with features of ß-thalassemia intermedia of various clinical severity. The disease mechanisms may involve duplicated α-globin genes, mosaic partial Uniparental Isodisomy of chromosome 11p15.4 where the HBB gene is located or haplo-insufficiency of a non-linked gene SUPT5H on chromosome 19q, first described in two Dutch families with ß-thalassemia trait without variants in the HBB gene.

Calibration by commutable control materials is able to reduce inter-method differences of current high-performance methods for HbA2.

BACKGROUND: Most of the current methods used for the determination of HbA2 seem not well aligned. A comparison among the best performing techniques and the commutability of some control materials currently available and under development has been evaluated. METHODS: Forty blood samples were analyzed in duplicate over two separate days by different HPLC and capillary electrophoresis systems. The commutabilities of different control materials (NIBSC WHO reagent, Bio-Rad Lyphochek, and home prepared lyophilized controls RP1-3) have been assessed by analyzing the controls in quadruplicate over two consecutive days together with the blood samples. RESULTS: The mean within-run imprecision of HbA2 measurement on blood samples (CV, %) was between 0.6% and 10.1% for HbA2 values <3.5%, and between 1.1 and 3.1 for HbA2≥3.5%. The different methods were highly correlated (r between 0.9941 and 0.9995) although biased each other. The NIBSC WHO reagent was found not commutable in 15 over 28 comparisons, the Lyphochek 2 in 18/28, and RP3 in 4/28. Recalibration of all methods by RP1 and RP2 materials was able to reduce the overall variability from 6.8% to 3.4% at HbA2≤3.0% and from 6.7% to 3.0% at HbA2≥4.6%. CONCLUSION: The use of adequate commutable control materials as calibrators may reduce the inter-method variability of routine methods to an extent closer to the current analytical goals of bias based on biological variability.

Melanocortin 1 receptor (MC1R) variants in high melanoma risk patients are associated with specific dermoscopic ABCD features.

Individuals with two red hair colour (RHC)-MC1R genetic variants have light skin and blond/reddish hair and, in comparison with those without such alleles, are at an increased risk of developing melanoma. Our study investigated the association of RHC variants and the Total Dermo-scopy Score (TDS), and the items that make up the TDS, in those with atypical naevi and melanomas from high risk melanoma patients. Eight hundred and seventy-six atypical naevi and 21 melanomas were scored according to the TDS system and MC1R polymorphisms were determined. Analyses revealed that several TDS items including pigment network, dark-brown colour and streaks were more frequently observed in atypical naevi from individuals without RHC variants, while structureless areas were more often observed in individuals with two RHC variants. Finally, no significant difference in TDS was detected in atypical naevi from individuals with two RHC variants compared to those without RHC. Clinicians should be aware of a different dermoscopic naevus pheno-type in patients with light blond or RHC MC1R variants.

Increased risk of cancer other than melanoma in CDKN2A founder mutation (p16-Leiden)-positive melanoma families.

PURPOSE: We report the largest study to date analyzing the risk of cancers other than melanoma in melanoma families positive for the same CDKN2A mutation. EXPERIMENTAL DESIGN: We studied family members of 22 families positive for the p16-Leiden founder mutation who had attended a surveillance clinic or were their close relatives. Within this cohort, observed and expected rates of cancer were computed by mutation status consisting of 221 (proven plus obligate) carriers, 639 (proven plus obligate) noncarriers, and 668 first-degree relatives whose carrier risk was estimated from the relationship to known carriers and the age and melanoma status of that person and their relatives. RESULTS: Our analysis shows a relative risk (RR) of cancer other than melanoma and nonmelanoma skin cancer of 4.4 [95% confidence interval (95% CI), 3.3-5.6], predominantly attributable to the increased risk for pancreatic cancer (RR, 46.6; 95% CI, 24.7-76.4), but also for other cancers. We provide substantial proof for pancreatic cancer being a key component of the p16-Leiden phenotype. Inclusion of this cancer in a penetrance analysis leads to an estimated RR of pancreatic cancer for mutation carriers of 47.8 (95% CI, 28.4-74.7). CONCLUSIONS: This study shows clear evidence of increased risk of cancers other than melanoma in CDKN2A families carrying the p16-Leiden mutation. Further research is necessary to determine if similar risks apply to families with CDKN2A mutations other than p16-Leiden.

Genetic testing in familial melanoma: uptake and implications.

OBJECTIVE: We report on the uptake and psychological impact of p16-Leiden genetic testing to contribute to a greater understanding of counseling melanoma families. METHODS: Within a defined research setting, genetic counseling and testing were offered to members of p16-Leiden-positive melanoma pedigrees, at risk of carrying a gene defect associated with an increased risk of melanoma and pancreatic cancer. RESULTS: One hundred and eighty-four individuals sought counseling, of which 141 (77%) opted for genetic testing. Uptake of genetic counseling and testing, and psychological motivation was evaluated in 94 (57%) individuals. Higher pre-test risk of carrying the mutation and older age proved significantly predictive for counseling uptake. Age was predictive for test acceptance, whereas fearful test expectancies predicted test decline. Counselees had lower distress levels than those reported in other oncogenetic testing settings. CONCLUSION: We are the first to report on genetic testing for familial melanoma. Following the first counseling session, we found a relatively high uptake rate for p16-Leiden testing and no clinically worrisome levels of distress.

A comparison of CDKN2A mutation detection within the Melanoma Genetics Consortium (GenoMEL).

CDKN2A is the major melanoma susceptibility gene so far identified, but only 40% of three or more case families have identified mutations. A comparison of mutation detection rates was carried out by "blind" exchange of samples across GenoMEL, the Melanoma Genetics Consortium, to establish the false negative detection rates. Denaturing high performance liquid chromatography (DHPLC) screening results from 451 samples were compared to screening data from nine research groups in which the initial mutation screen had been done predominantly by sequencing. Three samples with mutations identified at the local centres were not detected by the DHPLC screen. No additional mutations were detected by DHPLC. Mutation detection across groups within GenoMEL is carried out to a consistently high standard. The relatively low rate of CDKN2A mutation detection is not due to failure to detect mutations and implies the existence of other high penetrance melanoma susceptibility genes.

From sporadic atypical nevi to familial melanoma: risk analysis for melanoma in sporadic atypical nevus patients.

BACKGROUND: Atypical nevi (AN), present in either a familial or a sporadic setting, are strong indicators of increased melanoma risk. OBJECTIVE: To estimate the extent of this risk and the extent of reclassification of sporadic to familial cases during follow-up. METHODS: We studied 167 sporadic patients with AN (>or=5). At the end of follow-up we updated the family history regarding melanoma and performed germline mutation analysis of the known melanoma susceptibility genes. RESULTS: We found a relative risk for melanoma of 46.1 (95% confidence interval 21.0-87.5). Six of 167 patients were carriers of a CDKN2A mutation. At the end of follow-up, 10 of 136 patients with sporadic AN reported being a member of a melanoma family. LIMITATIONS: This study was conducted in an area with a founder mutation in many of its melanoma families; therefore the results may not be applicable to other populations. CONCLUSION: We report a high relative risk of 46.1 of melanoma development in patients with sporadic AN. A significant proportion of this Dutch cohort reported additional cases in their families over time.

A mutation hotspot at the p14ARF splice site.

Germline mutations of CDKN2A that affect the p16INK4a transcript have been identified in numerous melanoma pedigrees worldwide. In the UK, over 50% of pedigrees with three or more cases of melanoma have been found to carry mutations of CDKN2A. Mutations that affect p14ARF exon 1beta exclusively are very rare. This has led to the suggestion that it is p16INK4a and not p14ARF that plays the critical role in melanoma predisposition. We report the identification of a cluster of five different germline mutations at the p14ARF exon 1beta splice donor site in melanoma pedigrees. All the five splice site variants showed evidence of being causal mutations. Three of the variants were demonstrated to result in aberrant splicing of the p14ARF mRNA, confirming their role in melanoma predisposition. No other point mutations were identified in the coding region of p14ARF. The p14ARF transcript of CDKN2A is clearly important in disease predisposition in a subset of melanoma pedigrees. Curiously, the only mutations so far reported to affect p14ARF exon 1beta exclusively have been knockout mutations. Further investigation into the spectrum of mutations observed in this gene may help clarify the exact role of p14ARF in melanoma predisposition.