When to suspect infantile hypercalcemia-1?

PURPOSE: The screening test to suspect infantile hypercalcemia-1 (HCINF1) is the measure of 25(OH)D3/24,25(OH)2D3 ratio at mass spectroscopy (MS). When the ratio is > 80, the gold standard for the diagnosis is genetic analysis. Given its limited availability, MS may not represent a screening test and most cases of HCINF1 remain undiagnosed. Aim of the study is to identify cut-offs of serum calcium and PTH useful to suspect patients with HCINF1. METHODS: We compared the levels of total serum calcium and PTH of 6 patients with HCINF1 harboring biallelic CYP24A1 pathogenic variants with 3 different control groups: (1) 12 subjects wild type for CYP24A1; (2) 12 subjects matched for age and sex; (3) 12 subjects matched for vitamin D levels. We validated the cut-offs, testing the number of adult patients affected by HCINF1 reported in the literature that could be identified using these cut-offs. RESULTS: A serum calcium level > 9.6 mg/dL showed the highest sensitivity (100%) and specificity (91%) in the comparison between homozygous and wild-type subjects. A serum PTH index < 0.315 showed the highest sensitivity (100%) and specificity (83.3%). A serum calcium level > 9.6 mg/dL was able to identify all adult HCINF1 patients whereas a PTH ratio < 0.315 identified 89.8% of the cases. Superimposable results were obtained using the other control groups. CONCLUSION: Patients with serum calcium levels higher than 9.6 mg/dL and a PTH index lower than 0.315 are likely to be affected by HCINF1. Their diagnosis may be confirmed using MS and genetic analysis.

Clinical and molecular characteristics of two Italian kindreds with hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome.

PURPOSE: Hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome, also known as Barakat syndrome, is a rare autosomal dominant disease characterized by the triad of hypoparathyroidism, deafness, and renal abnormalities. The disorder is caused by the haploinsufficiency of the zinc finger transcription factor GATA3 and exhibits a great clinical variability with an age-dependent penetrance of each feature. We report two unrelated kindreds whose probands were referred to our outpatient clinic for further evaluation of hypoparathyroidism. METHODS: The proband of family 1, a 17-year-old boy, was referred for severe hypocalcemia (5.9 mg/dL) incidentally detected at routine blood tests. Abdomen ultrasound showed bilateral renal cysts. The audiometric evaluation revealed the presence of bilateral moderate hearing loss although the patient could communicate without any problem. Conversely, the proband of family 2, a 19-year-old man, had severe symptomatic hypocalcemia complicated by epileptic seizure at the age of 14 years; his past medical history was remarkable for right nephrectomy at the age of 4 months due to multicystic renal disease and bilateral hearing loss diagnosed at the age of 18 years. RESULTS: Based on clinical, biochemical, and radiologic data, HDR syndrome was suspected and genetic analysis of the GATA3 gene revealed the presence of two pathogenetic variants in exon 3, c.404dupC and c.431dupG, in the proband of family 1 and 2, respectively. CONCLUSION: HDR syndrome is a rare cause of hypoparathyroidism and must be excluded in all patients with apparently idiopathic hypoparathyroidism. A correct diagnosis is of great importance for early detection of other HDR-related features and genetic counseling.

Disorders of sexual development with XY karyotype and female phenotype: clinical findings and genetic background in a cohort from a single centre.

PURPOSE: 46, XY disorders (or differences) of sex development (DSD) are a group of clinical conditions with variable genetic background; correct diagnosis is often difficult, but it permits to optimize the management. The aim of this study is to identify clinical and genetics features of a group of women with 46, XY DSD to define some issues characterizing people with 46, XY DSD in Italy. METHODS: Retrospective analysis of girls and women with 46, XY DSD and female phenotype evaluated between year 2000 and 2016, performed by anonymised database, focusing on the clinical features and management, including presentation, first diagnostic suspect, gonadal surgery and molecular diagnostic delay. RESULTS: A total of 84 records were collected (mean age at clinical presentation: 9.1 ± 7.9 years; mean age at definitive diagnosis: 20.1 ± 15.0 years). Complete androgen insensitivity syndrome was the most common diagnosis (60%). Only 12 patients (14.3%) did not receive a molecular diagnosis. Early misdiagnoses frequently occurred; diagnostic delay was 10.2 ± 11.2 years, being reduced in patients presenting from 2007 to 2016. The discordance between genotypic and phenotypic sex during pregnancy or at birth determined early reason for referral in a considerable percentage (4.9%). CONCLUSION: Misdiagnosis and long diagnostic delays are present in females with 46, XY DSD in Italy, but the new genetic techniques permit faster right diagnoses in the last years. The centralization in dedicated third level units permits to reduce the number of patients without a molecular diagnosis, allowing better clinical management and appropriate genetic counselling.

Correction to: Whole exome sequencing in familial isolated primary hyperparathyroidism.

Unfortunately, the 13th author name has been published incorrectly in the original publication.

PROMs in post-mastectomy care: Patient self-reports (BREAST-Q™) as a powerful instrument to personalize medical services.

One of the goals of immediate breast reconstruction (IBR) is to satisfy the patient's outcome. Recent studies therefore tended to focus on the patient's perception of the care and on the impact on quality of life using patients-reported-outcome-measures (PROMs), able to measure the health status directly without the clinician's interposition. We present a preliminary prospective study on 333 patients who underwent mastectomy with IBR in a two-year period, in a single Italian centre, using a dedicated PROMs, the BREAST-Q™, to determine the patient's satisfaction. We studied two groups of IBR: Group A (two-step with tissue-expander) and Group B (one-step: prosthesis/mesh) and conducted a pre- and post-operative comparison for each group to evaluate score-gain over time, and a group-score comparison to determine whether differences were significant between reconstruction types. Two-hundred-and-nine were actually enrolled and 132 completed all the questionnaires. The response rate was 62.8% and the compliance rate (completion of all the questionnaires) was 63.1%. In both groups all the analyzed domains worsened comparing the pre and post-operative period; the differences were statistically significant only for physical and sexual-wellbeing. In the comparison between the two groups, none of the detected differences reached the statistical significance. According to our experience, we can state that PROMs could improve the health concept redefining the variables to be monitored even if data is still insufficient to draw any definitive conclusion. PROMs can help surgeons and patients decide the most appropriate surgery for a particular patient-profile and to identify those who require further support.

Whole exome sequencing in familial isolated primary hyperparathyroidism.

PURPOSE: Familial isolated hyperparathyroidism (FIHP) is a rare inherited disease accounting for 1% of all cases of primary hyperparathyroidism (PHPT). It is genetically heterogeneous being associated with mutations in different genes, including MEN1, CDC73, CASR, and recently GCM2. The aim of the study was to further investigate the molecular pathogenesis in Italian FIHP kindreds. METHODS: We used whole exome sequencing (WES) in the probands of seven unrelated FIHP kindreds. We carried out a separate family-based exome analysis in a large family characterized by the co-occurrence of PHPT with multiple tumors apparently unrelated to the disease. Selected variants were also screened in 18 additional FIHP kindreds. The clinical, biochemical, and pathological characteristics of the families were also investigated. RESULTS: Three different variants in GCM2 gene were found in two families, but only one (p.Tyr394Ser), already been shown to be pathogenic in vitro, segregated with the disease. Six probands carried seven heterozygous missense mutations segregating with the disease in the FAT3, PARK2, HDAC4, ITPR2 and TBCE genes. A genetic variant in the APC gene co-segregating with PHPT (p.Val530Ala) was detected in a family whose affected relatives had additional tumors, including colonic polyposis. CONCLUSION: We confirm the role of GCM2 germline mutations in the pathogenesis of FIHP, although at a lower rate than in the previous WES study. Further studies are needed to establish the prevalence and the role in the predisposition to FIHP of the novel variants in additional genes.

Germline investigation in male breast cancer of DNA repair genes by next-generation sequencing.

PURPOSE: In order to better define the breast cancer (BC) genetic risk factors in men, a germline investigation was carried out on 81 Male BC cases by screening the 24 genes involved in BC predisposition, genome stability maintenance and DNA repair mechanisms by next-generation sequencing. METHODS: Germline DNAs were tested in a custom multi-gene panel focused on all coding exons and exon-intron boundaries of 24 selected genes using two amplicon-based assays on PGM-Ion Torrent (ThermoFisher Scientific) and MiSeq (Illumina) platforms. All variants were recorded and classified by using a custom pipeline. RESULTS: Clinical pathological data and the family history of 81 Male BC cases were gathered and analysed, revealing the average age of onset to be 61.3 years old and that in 35 cases there was a family history of BC. Our genetic screening allowed us to identify a germline mutation in 22 patients (23%) in 4 genes: BRCA2, BRIP1, MUTYH and PMS2. Moreover, 12 variants of unknown clinical significance (VUS) in 9 genes (BARD1, BRCA1, BRIP1, CHEK2, ERCC1, NBN, PALB2, PMS1, RAD50) were predicted as potentially pathogenic by in silico analysis bringing the mutation detection rate up to 40%. CONCLUSION: As expected, a positive family history is a strong predictor of germline BRCA2 mutations in male BC. Understanding the potential pathogenicity of VUS represents an extremely urgent need for the management of BC risk in Male BC cases and their own families.

Association of SULT1A1 Arg²¹³His polymorphism with male breast cancer risk: results from a multicenter study in Italy.

Male breast cancer (MBC) is rare and poorly understood. Like female breast cancer (FBC), MBCs are highly sensitive to hormonal changes, and hyperestrogenism, specifically, represents a major risk factor for MBC. MBC is considered similar to late-onset, post-menopausal estrogen/progesteron receptors positive FBC (ER+/PR+). Sulfotransferase 1A1 (SULT1A1) is an enzyme involved in the metabolism of estrogens. Recently, SULT1A1 common functional polymorphism Arg(213)His (638G>A) variant has been found to be associated with increased breast cancer (BC) risk, particularly in post-menopausal women. For this reason, we decided to explore whether SULT1A1 Arg(213)His could exert an effect on MBC development. The primary aim of this study was to evaluate the influence of the SULT1A1 Arg(213)His polymorphism on MBC risk. The secondary aim was to investigate possible associations with relevant clinical-pathologic features of MBC. A total of 394 MBC cases and 786 healthy male controls were genotyped for SULT1A1 Arg(213)His polymorphism by PCR-RFLP and high-resolution melting analysis. All MBC cases were characterized for relevant clinical-pathologic features. A significant difference in the distribution of SULT1A1 Arg(213)His genotypes was found between MBC cases and controls (P < 0.0001). The analysis of genotype-specific risk showed a significant increased MBC risk in individuals with G/A (OR 1.97, 95% CI 1.50-2.59; P < 0.0001) and A/A (OR 3.09, 95% CI 1.83-5.23; P < 0.0001) genotypes in comparison to wild-type genotype, under co-dominant model. A significant association between SULT1A1 risk genotypes and HER2 status emerged. Results indicate that SULT1A1 Arg(213)His may act as a low-penetrance risk allele for developing MBC and could be associated with a specific tumor subtype associated with HER2 overexpression.

Performance of BOADICEA and BRCAPRO genetic models and of empirical criteria based on cancer family history for predicting BRCA mutation carrier probabilities: a retrospective study in a sample of Italian cancer genetics clinics.

PURPOSE: To evaluate in current practice the performance of BOADICEA and BRCAPRO risk models and empirical criteria based on cancer family history for the selection of individuals for BRCA genetic testing. PATIENTS AND METHODS: The probability of BRCA mutation according to the three tools was retrospectively estimated in 918 index cases consecutively undergone BRCA testing at 15 Italian cancer genetics clinics between 2006 and 2008. RESULTS: 179 of 918 cases (19.5%) carried BRCA mutations. With the strict use of the criteria based on cancer family history 173 BRCA (21.9%) mutations would have been detected in 789 individuals. At the commonly used 10% threshold of BRCA mutation carrier probability, the genetic models showed a similar performance [PPV (38% and 37%), sensitivity (76% and 77%) and specificity (70% and 69%)]. Their strict use would have avoided around 60% of the tests but would have missed approximately 1 every 4 carriers. CONCLUSION: Our data highlight the complexity of BRCA testing referral in routine practice and question the strict use of genetic models for BRCA risk assessment.

Evaluation of the XRCC1 gene as a phenotypic modifier in BRCA1/2 mutation carriers. Results from the consortium of investigators of modifiers of BRCA1/BRCA2.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in genes involved in DNA repair are good candidates to be tested as phenotypic modifiers for carriers of mutations in the high-risk susceptibility genes BRCA1 and BRCA2. The base excision repair (BER) pathway could be particularly interesting given the relation of synthetic lethality that exists between one of the components of the pathway, PARP1, and both BRCA1 and BRCA2. In this study, we have evaluated the XRCC1 gene that participates in the BER pathway, as phenotypic modifier of BRCA1 and BRCA2. METHODS: Three common SNPs in the gene, c.-77C>T (rs3213245) p.Arg280His (rs25489) and p.Gln399Arg (rs25487) were analysed in a series of 701 BRCA1 and 576 BRCA2 mutation carriers. RESULTS: An association was observed between p.Arg280His-rs25489 and breast cancer risk for BRCA2 mutation carriers, with rare homozygotes at increased risk relative to common homozygotes (hazard ratio: 22.3, 95% confidence interval: 14.3-34, P<0.001). This association was further tested in a second series of 4480 BRCA1 and 3016 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. CONCLUSIONS AND INTERPRETATION: No evidence of association was found when the larger series was analysed which lead us to conclude that none of the three SNPs are significant modifiers of breast cancer risk for mutation carriers.

Evaluation of a candidate breast cancer associated SNP in ERCC4 as a risk modifier in BRCA1 and BRCA2 mutation carriers. Results from the Consortium of Investigators of Modifiers of BRCA1/BRCA2 (CIMBA).

BACKGROUND: In this study we aimed to evaluate the role of a SNP in intron 1 of the ERCC4 gene (rs744154), previously reported to be associated with a reduced risk of breast cancer in the general population, as a breast cancer risk modifier in BRCA1 and BRCA2 mutation carriers. METHODS: We have genotyped rs744154 in 9408 BRCA1 and 5632 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and assessed its association with breast cancer risk using a retrospective weighted cohort approach. RESULTS: We found no evidence of association with breast cancer risk for BRCA1 (per-allele HR: 0.98, 95% CI: 0.93-1.04, P = 0.5) or BRCA2 (per-allele HR: 0.97, 95% CI: 0.89-1.06, P = 0.5) mutation carriers. CONCLUSION: This SNP is not a significant modifier of breast cancer risk for mutation carriers, though weak associations cannot be ruled out.

Two mutations of BRCA2 gene at exon and splicing site in a woman who underwent oncogenetic counseling.

BACKGROUND: Although most BRCA sequence variants are clearly deleterious and unequivocally pathogenetic, several are still classified as variants of unknown significance. PATIENTS AND METHODS: We followed families undergoing oncogenetic counseling from risk identification to risk definition by genetic testing and risk management. RESULTS: We identified two germline mutations in the BRCA2 gene in a woman with breast and ovarian cancer. One sequence alteration was 859/G>A in exon 7 (V211I). The other second sequence alteration (IVS13-2A>T) affected the splicing site in intron 13. The latter alteration is not yet listed in the Breast Cancer Information Core database. RT-PCR resulted in transcription of a sequence lacking exon 7 and a subsequent anomalous stop codon in exon 9 thereby confirming altered messenger RNA (mRNA) maturation. Amplification of the mutation in intron 13 resulted in transcription of a sequence lacking exon 14 and an anomalous stop codon in exon 15 thereby confirming altered mRNA maturation. Both mutations led to a truncated BRCA2 protein in its carboxy-terminal region. CONCLUSION: The two BRCA2 mutations identified affect mRNA splicing fidelity and play a pathogenetic role in breast and ovarian cancer.

Reconstructing the genealogy of a BRCA1 founder mutation by phylogenetic analysis.

Estimating the age of founder mutations may contribute to improve our knowledge of population genetics and evolutionary history of diseases. Previous haplotype analysis suggested that the BRCA1*1499insA mutation was a founder allele, probably originated in Tuscany (Italy). Here, we collected additional pedigrees carrying this mutation, and applied a phylogenetic method for estimating mutation age. A chromosome segment of about 25 cM, including 37 short tandem repeats (STRs) on both sides of the BRCA1 gene (DeCode map), was typed in 50 subjects (28 mutation carriers) from 14 unrelated families. The time to the most recent common ancestor (MRCA) of the mutation carriers was estimated by the length of the shared haplotype between all possible pairs of individuals. A function relating the length of the shared haplotype to the time to the MRCA was obtained by a computer simulation. This approach gives results comparable with those of other existing mutation-dating methods, but does not depend explicitly on population-specific parameters such as allele frequencies, provides narrower confidence intervals (CI), and allows one to build an extended genealogical tree of all mutation carriers. The 1499insA mutation shared by the investigated subjects was estimated to be present in an individual living about 30 generations ago (95% CL 22-56), or 750 years (95% CL 550-1,400).

Methyl group metabolism gene polymorphisms as modifier of breast cancer risk in Italian BRCA1/2 carriers.

BRCA1 and 2 are major cancer susceptibility genes but their penetrance is highly variable. The folate metabolism plays an important role in DNA methylation and its alterated metabolism is associated with cancer risk. The role of allele variants 677T and 1298C (MTHFR gene) and 2756G (MS gene) has been investigated as potentially modifying factors of BRCA gene penetrance, evaluated as age at first diagnosis of cancer, in 484 BRCA1/BRCA2 carriers and in 108 sporadic breast cancer cases as a control group. The genotype analysis has been performed by means of PCR/RFLP's. The analysis of association between a particular genotype and disease risk was performed using Cox Regression with time to breast or ovarian cancer onset as the end-point. The presence of 677T allele confers an increased risk of breast cancer in BRCA1 carriers (P = 0.007) and the presence of 1298C allele confers an increased risk of breast cancer in sporadic cases (P = 0.015).

Genetic alterations in hereditary breast cancer.

Genetic linkage studies have led to the identification of highly penetrant genes as the possible cause of inherited cancer risk in many cancer-prone families. Most women with a family history of breast/ovarian cancer have tumors characterized by alterations in particular genes, mainly BRCA1 and BRCA2, but also CHK2, ATM, STK11 and others. This paper examines the BRCA1 and BRCA2 genes, focusing on the Italian pattern of mutations. The function of these two genes, classified as tumor suppressors, is linked with key metabolic mechanisms such as DNA damage repair, regulation of gene expression and cell cycle control. The pathological BRCA allelic variants may cause alteration of protein function, transcriptional activity and DNA repair; accumulation of the defects leads to widespread chromosome instability that may be directly responsible for cancer formation. In fact, mutations in BRCA1 and BRCA2, conferring a highly increased susceptibility to breast and ovarian cancer, do not lead to cancer by themselves. The current consensus is that these are 'caretaker' genes, which, when inactivated, allow other genetic defects to accumulate. The nature of these other molecular events may define the pathway through which BRCA1 and BRCA2 act. The BRCA mutation spectrum is complex, and the significance of most nucleotide alterations is difficult to understand. Moreover, the mutation pattern seems to be related to ethnicity. The Italian Consortium of Hereditary Breast and Ovarian Cancer has reviewed 1758 families; 23% have been found to be carriers of pathogenetic mutations in BRCA1 or BRCA2. Founder mutations have been described in geographically restricted areas of Italy; a regional founder effect has been demonstrated in Italy for the mutations BRCA1 5083del19 and BRCA2 8765delAG, and a probable new founder mutation has been characterized in Tuscany. The presence of founder mutations has practical implications for genetic testing.

The Italian multi-centre project on evaluation of MRI and other imaging modalities in early detection of breast cancer in subjects at high genetic risk.

This report presents the preliminary results of the first phase (21 months) of a multi-centre, non-randomised, prospective study, aimed at evaluating the effectiveness of contrast-enhanced magnetic resonance imaging (MRI), X-ray mammography (XM) and ultrasound (US) in early diagnosis of breast cancer (BC) in subjects at high genetic risk. This Italian national trial (coordinated by the Istituto Superiore di Sanità, Rome) so far recruited 105 women (mean age 46.0 years; median age 51.0; age range 25-77 years), who were either proven BRCA1 or BRCA2 mutation carriers or had a 1 in 2 probability of being carriers (40/105 with a previous personal history of BC). Eight cases of breast carcinomas were detected in the trial (mean age 55.3 years, median age 52.5; age range 35-70 years; five with previous personal history of BC). All trial-detected BC cases (8/8) were identified by MRI, while XM and US correctly classified only one. MRI had one false positive case, XM and US none. Seven "MRI-only" detected cancers (4 invasive, 3 in situ) occurred in both pre- (n = 2) and post-menopausal (n = 5) women. With respect to the current XM screening programmes addressed to women in the age range 50-69 years, the global incidence of BC in the trial (7.6%) was over ten-fold higher. The cost per "MRI-only" detected cancer in this particular category of subjects at high genetic risk was substantially lower than that of an XM-detected cancer in the general women population. These preliminary results confirmed that MRI is a very useful tool to screen subjects at high genetic risk for breast carcinoma, not only in pre-, but also in post-menopausal age, with a low probability of false positive cases.

Breast MR imaging screening in eight women proved or suspected to be carriers of BRCA1&2 gene mutations.

The aim of this study was to evaluate the efficacy of MR imaging for the early detection of breast tumor in women at high genetic risk compared to conventional strategies such as ultrasonography and mammography. This study included 8 women, 5 of which had undergone surgery for breast cancer. BRCA germ line mutations were detected in 7 women, one patient was enrolled for more than 50% probability to be carrier of BRCA mutation. RM imaging screening was negative in 7 patients and strongly indicative of a malignant lesion in one. The gold standard was surgery for the suspicious cases and follow-up with clinical examination and conventional imaging every six months for the others. MR imaging proved itself to be a reliable technique in familial breast cancer high risk women.