Opt-in for secondary findings as part of diagnostic whole-exome sequencing: Real-life experience from an international diagnostic laboratory.

BACKGROUND: Discussion about the risks and benefits of offering secondary findings as part of genome-wide diagnostics lacks real-life data. We studied the opt-in decisions of patients/families referred to whole exome study (WES) in Blueprint Genetics (BpG), a genetic testing company with customers in over 70 countries to receive secondary findings. Based on the American College of Medical Genetics (ACMG) recommendations for reporting secondary findings, BpG offered testing of specific actionable genes without additional charge for specimens submitted to WES diagnostics. METHODS: Individuals could opt-in for a secondary findings analysis by using a separate electronic consent form. Data from BpG database of electronic consent forms was used for the analysis. RESULTS: During the selected study period there were 3263 WES referrals, from which 2012 were index patients. About half of the individuals (50.4%) opted in to receiving secondary findings. Of patients who opted in, a secondary finding was detected for 2.7%, similar to other studies. We detected huge differences relating to opt-in between individuals from different countries; for instance, 90% of the 41 patients and their family members in Romania opted to receive secondary findings, while none of the 98 patients in Luxembourg chose that option. CONCLUSION: Differences between sexes or between children and adults were small. This data offers one view to the interest of patients and family members to opt in to receiving secondary findings. Research is needed to understand the influence of factors like age, education etc. and possible participation in pre-test counseling to receiving/not receiving secondary findings.

Phenotype expansion and neurological manifestations of neurobehavioural disease caused by a variant in RFX7.

The RFX7 gene is one of eight genes within the regulatory factor X family. RFX7 is highly expressed in the brain and plays an important role in cell maturation and differentiation. It has only recently been implicated in disease in humans. Reports from 15 individuals have described RFX-associated phenotype as a neurobehavioural disease, manifesting primarily with global developmental delay and intellectual disability. Autism spectrum disorder and attention deficit hyperactivity disorder have also been described in some children. Here we report a case of a 19-month-old with a de novo missense variant in RFX7 resulting in severe global developmental delay including significant speech delay, microcephaly, dyskinetic movements, and failure to thrive. This is the first association between variants in RFX7 and failure to thrive, expanding the phenotype of this newly described gene. In this report we will also show RFX7 associated progressive central nervous system involvement through serial brain imaging.

Infantile-Onset Charcot-Marie-Tooth Disease With Pyramidal Features and White Matter Abnormalities Due to a De novo MORC2 Gene Variant: A Case Report and Brief Review of the Literature.

Background: Charcot-Marie-Tooth (CMT) is the most frequent group of inherited neuropathies and includes several heterogeneous phenotypes. Over 80 causative genes have been described so far. Variants in the microrchidia family CW-type zinc finger 2 (MORC2) gene have been described in several axonal polyneuropathy (CMT2) patients with childhood or adult onset. Occasionally more complex phenotypes with delayed milestones, severe hypotonia, intellectual disability, dystonic postures, pyramidal signs, and neuroimaging abnormalities have been reported. Case Presentation: We report on a patient with a de novo MORC2 gene variant (c.1181A>G p.Tyr394Cys) with a history of developmental delay, axial hypotonia, progressive gait disorder with dystonic features, and intentional tremor. At the age of 8 years, he showed bilateral pyramidal signs (clonus, increased tendon reflexes, and Babinski sign) and bilateral pes cavus. The first neuroimaging performed at the age of 3 years demonstrated white matter abnormalities in the posterior periventricular zone, in the frontal lobes bilaterally and at the midbrain, stable during childhood and adolescence. Nerve conduction studies (NCS) were negative until the age of 15 years, when a sensory axonal neuropathy appeared. The association between pyramidal signs and neuropathy due to the MORC2 gene variant is increasingly being highlighted, although a neuroradiological correlate is evident only in about half of the cases. Longitudinal nerve conduction velocity (NCV) are helpful to identify late-onset features and provide useful information for diagnosis in patients with rare neurogenetic disorders. Conclusions: Characterization of complex neurological disorders is important to delineate the expanding phenotypic spectrum of MORC2-related disease, to confirm if possible the pathogenicity of the variants and to deepen the genotype-phenotype correlation.

EGFR gene copy number predicts response to anti-EGFR treatment in RAS wild type and RAS/BRAF/PIK3CA wild type metastatic colorectal cancer.

Anti-EGFR antibodies are used for the treatment of RAS wild type metastatic colorectal cancer. We previously showed that EGFR gene copy number (GCN) predicts response to anti-EGFR therapy in KRAS exon 2 wild type metastatic colorectal cancer. The aim of our study was to analyse the predictive role of EGFR GCN in RAS/BRAF/PIK3CA wild type metastatic colorectal cancer. The material included 102 patients with KRAS exon 2 wild type metastatic colorectal cancer treated with anti-EGFR ± cytotoxic therapy. Next generation sequencing was used for KRAS, NRAS, BRAF and PIK3CA gene mutation analyses. EGFR GCN was analysed by EGFR immunohistochemistry guided automated silver in situ hybridisation. Increased EGFR GCN (≥4.0) predicted a better response and prolonged progression free survival in anti-EGFR treated RAS/BRAF/PIK3CA wild type patients (Log-rank test, p = 0.0004). In contrast, survival of RAS/BRAF/PIK3CA wild type, EGFR GCN below 4.0 patients did not differ from patients with mutant RAS, BRAF or PIK3CA. Our study indicates that EGFR GCN predicts anti-EGFR treatment efficacy in patients with RAS/BRAF/PIK3CA wt metastatic CRC. Tumours with EGFR GCN below 4.0 appear to be as refractory to anti-EGFR treatment as tumours with mutation in any of the RAS/RAF/PIK3CA pathway genes.

Study of p53 gene mutations and placental expression in recurrent miscarriage cases.

This study aimed to investigate the role of p53 in early human development by screening patients with recurrent miscarriages (RM) for mutations in the p53 gene and by studying p53 expression in placental tissue. A total of 46 women with RM and 191 control women were included in the study. A sample was also obtained from 40 male partners of RM patients. The samples were screened for p53 sequence variations using denaturing high-performance liquid chromatography, sequencing and allele-specific polymerase chain reaction. Placental tissue was available from 19 miscarriages. p53 expression in placental tissue was studied by immunohistochemical staining. The C11992A polymorphism in p53 was found to be associated with RM in Finnish patients. The C/A or A/A genotype was detected in 32.6% of the women with RM and in 18.9% of the controls (P = 0.0414, odds ratio 2.083, confidence interval 1.018-4.259). The results suggest that women carrying the C/A or A/A genotype have a two-fold higher risk for RM than women with the C/C genotype. Further studies are, however, necessary to define whether the intronic polymorphism has functional consequences. The immunohistochemical staining of placental tissues revealed no abnormal p53 expression patterns in the samples studied.

Do mitochondrial mutations cause recurrent miscarriage?

The cause of recurrent miscarriage (RM) can be identified in approximately 50% of cases, whereas in others, unknown genetic factors are actively being sought. As mitochondrial functions, and therefore also the mitochondrial genome [mitochondrial DNA (mtDNA)], have an important role in human development, through ATP production and participation in apoptosis, we aimed to study the role of mtDNA variations in RM. We screened 48 women with RM and 48 age-matched control women for heteroplasmic mitochondrial mutations using denaturing high performance liquid chromatography, a sensitive method that can detect approximately 5% heteroplasmy. As a result, we detected a heteroplasmic mtDNA variation in 13 RM women (27%) and in 9 control women (19%). Seven synonymous and five non-synonymous changes were detected within coding regions. In addition, seven heteroplasmic variations were detected within the non-coding control region. We were also able to show the presence of the variations in eight placental samples from three heteroplasmic women. In three of these cases, the proportion of variant mtDNA was higher in the placenta compared with that in the mother. We conclude that our sensitive methodology revealed a higher frequency of samples with heteroplasmic variations than expected in women with both RM and controls. However, no apparent increased frequency of heteroplasmic mtDNA variations or amounts of aberrant mtDNA was detected in the RM group. In addition, none of the detected variations were previously known to be pathogenic and therefore they are an unlikely cause of miscarriage.

Non-synonymous sequence variants within the oxygen-dependent degradation domain of the HIF1A gene are not associated with pre-eclampsia in the Finnish population.

BACKGROUND: Reduced placental perfusion predisposes to the maternal syndrome pre-eclampsia characterized by systemically reduced perfusion. Considerable data support the role of angiogenic factors in the development of the maternal syndrome. Hypoxia-inducible factor (HIF-1) mediates the cellular responses to hypoxia e.g. by promoting angiogenesis. METHODS: Here we studied whether two single nucleotide sequence variants, c.1744 C>T that changes residue 582 of HIF-1alpha from proline to serine (P582S) and c.1762 G>A that changes residue 588 of HIF-1alpha from alanine to threonine (A588T) in the exon 12 of the HIF1A gene, are associated with pre-eclampsia. We studied 108 women with pre-eclampsia in their first pregnancy, and 101 controls with normotensive pregnancies. Pre-eclampsia was defined as a blood pressure level of at least 140/90 mmHg in a woman who was normotensive before 20 weeks of gestation, and proteinuria at least of 0.3 g per 24-hour urine collection. The patients and controls were genotyped for variations in the exon 12 of HIF1A gene by sequencing RESULTS: The frequencies of the c.1744 C>T and c.1762G>A sequence variants were not significantly different between women with pre-eclamptic first pregnancies and women with normotensive pregnancies. In addition, two synonymous variants (c.1740G>A and c.1800A>T) were detected at comparable levels in the two groups. All variants were identified in the heterozygous form. CONCLUSION: The sequence variants in the exon 12 of the HIF1A gene were not associated with pre-eclampsia in the Finnish population.

Chorionic gonadotropin beta-gene variants are associated with recurrent miscarriage in two European populations.

CONTEXT: The incidence of recurrent miscarriage (RM) (>or=3 consecutive pregnancy losses) is estimated as 1-2% in fertile couples. Familial clustering of RM has suggested the contribution of a genetic component. OBJECTIVE: A low level of human chorionic gonadotropin (HCG) in maternal serum during the first trimester of the pregnancy is a clinically accepted risk factor for miscarriage. We sought to study whether variation in chorionic gonadotropin beta-subunit genes (CGBs) expressed in placenta may contribute to the risk of RM. DESIGN: Resequencing of CGB5 and CGB8, the two most actively transcribed loci of the four HCG beta-duplicate genes, was performed. SETTING: A case-control study involving two sample sets, from Estonia (n = 194) and Finland (n = 185), was performed. PATIENTS: RM patients (n = 184) and fertile controls (n = 195) participated in the study. RESULTS: From 71 identified variants in CGB5 and CGB8, 48 polymorphisms were novel. Significant protective effect was associated with two single nucleotide polymorphisms located at identical positions in intron 2 in both CGB5 [P = 0.007; odds ratio (OR) = 0.53] and CGB8 (P = 0.042; OR = 0.15), and with four CGB5 promoter variants (P < 0.03; OR = 0.54-0.58). The carriers of minor alleles had a reduced risk of RM. The haplotype structure of the CGB8 promoter was consistent with balancing selection; a rare mutation in CGB8 initiator element was detected only among patients (n = 3). In addition, three rare nonsynonymous substitutions were identified among RM cases as possible variants increasing the risk of recurrent pregnancy loss. CONCLUSION: The findings encourage studying the functional effect of the identified variants on CGB expression and HCG hormone activity to elucidate further the role of CGB variation in RM.

Sex chromosome characteristics and recurrent miscarriage.

OBJECTIVE: To investigate whether skewed X chromosome inactivation (XCI) and Y chromosome microdeletions are associated with recurrent miscarrige (RM). DESIGN: A retrospective study. SETTING: University hospital and genetic laboratory. PATIENT(S): Altogether, 46 women with a history of RM, defined as at least three miscarriages, and a control group of 95 women with no history of miscarriage were included in the XCI study. In the Y chromosome microdeletion study 40 male partners of women with RM were studied. INTERVENTION(S): Blood samples for DNA extraction. MAIN OUTCOME MEASURE(S): X chromosome inactivation patterns in the females were analyzed using a methylation-sensitive assay. The DNA from males was tested for Y chromosome microdeletions by analyzing 37 sequence tagged sites. RESULTS: Mildly skewed XCI (>85% inactivation of one allele) was detected in 4 of 43 (9.3%) patients, and 9 of 81 (11.1%) controls. Among these women, extremely skewed XCI (>90% inactivation of one allele) was detected in 2 of 43 (4.7%) patients, and 4 of 81 (4.9%) controls. No statistical differences could be shown between the groups. No microdeletions were found in the male partners. CONCLUSION(S): The frequency of both extremely and mildly skewed XCI was similar in patients and control women. Y chromosome microdeletions were not found in spouses of patients. Based on these results we conclude that skewed X inactivation and Y chromosome microdeletions are not associated with RM in our study couples.

Variations of the Amnionless gene in recurrent spontaneous abortions.

Recurrent spontaneous abortions (RSA) are estimated to affect 0.5-1% of couples trying to have a child. The causes of RSA are unknown in the majority of cases. This study aimed to determine whether homozygous mutations in the AMN gene in a fetus cause spontaneous abortions in humans, as they are known to cause spontaneous abortions in mice. The study was conducted by screening 40 couples and 5 women with three or more unexplained spontaneous abortions for heterozygous mutations in the AMN gene using denaturing high-performance liquid chromatography. Altogether, 3 exonic and 11 intronic sequence variations were found. There were no significant differences in the frequencies of the variations between the patients and a control group. One of the exonic variations was non-synonymous, and three of the variations may affect gene splicing. None of the putative phenotype-affecting variations were found in both partners in any couple. These results indicate that RSA in the couples studied cannot be explained by homozygous AMN mutations in the fetus. However, two couples had different, potentially deleterious variations in both partners. If these variations have a phenotypic effect, the RSA experienced by these couples may be caused by mutations in the AMN gene. In addition, birthplaces of the patients' ancestors revealed some clustering, suggesting that some patients may carry a founder mutation in another gene which may contribute to RSA.

Evaluation of RAD50 in familial breast cancer predisposition.

The genes predisposing to familial breast cancer are largely unknown, but 5 of the 6 known genes are involved in DNA damage repair. RAD50 is part of a highly conserved complex important in recognising, signalling and repairing DNA double-strand breaks. Recently, a truncating mutation in the RAD50 gene, 687delT, was identified in 2 Finnish breast cancer families. To evaluate the contribution of RAD50 to familial breast cancer, we screened the whole coding region for mutations in 435 UK and 46 Finnish familial breast cancer cases. We identified one truncating mutation, Q350X, in one UK family. We screened a further 544 Finnish familial breast cancer cases and 560 controls for the 687delT mutation, which was present in 3 cases (0.5%) and 1 control (0.2%). Neither Q350X nor 687delT segregated with cancer in the families in which they were identified. Functional analyses suggested that RAD50 687delT is a null allele as there was no detectable expression of the mutant protein. However, the wild-type allele was retained and expressed in breast tumors from mutation carriers. The abundance of the full-length RAD50 protein was reduced in carrier lymphoblastoid cells, suggesting a possible haploinsufficiency mechanism. These data indicate that RAD50 mutations are rare in familial breast cancer and either carry no, or a very small, increased risk of cancer. Altogether, these results suggest RAD50 can only be making a very minor contribution to familial breast cancer predisposition in UK and Finland.

Breast cancer patients with p53 Pro72 homozygous genotype have a poorer survival.

PURPOSE: The p53 R72P polymorphism has been suggested to play a role in many cancers, including breast cancer. Our aim was to evaluate association of R72P with breast cancer risk as well as histopathologic features of the breast tumors and survival. EXPERIMENTAL DESIGN: The germ line R72P genotype was defined among 939 Finnish familial and 888 unselected breast cancer patients and 736 healthy population controls. The clinical and biological variables were tested for association by univariate analysis and the effects of several variables on survival by Cox's proportional hazards regression model. RESULTS: The distribution of the genotypes was similar in all groups studied, suggesting no association with breast cancer risk. Unselected breast cancer patients with 72P homozygous genotype presented significantly more often with lobular carcinoma, whereas R72 allele carriers had a significantly higher frequency of ductal carcinomas (P = 0.004). No significant association with other histopathologic variables, like tumor grade, hormone receptor status (estrogen and progesterone receptors), or tumor-node-metastasis stage, was observed. Survival analysis showed that unselected breast cancer patients with 72P homozygous genotype had significantly poorer survival than patients with other genotypes (P = 0.003). This effect on survival was independent of p53 expression in the tumors and multivariate analysis showed that 72P homozygous genotype was overall an independent prognostic factor (risk ratio of death, 2.1; 95% confidence interval, 1.4-3.3; P = 0.001). CONCLUSIONS: These results suggest no effect of either R72P allele on breast cancer risk but a significantly reduced survival for 72P homozygous breast cancer patients. The finding of codon 72 genotype as an independent prognostic marker for breast cancer warrants further studies.