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Aims Cardiac amyloidosis remains a great challenge for the cardiologist. One of the three main aetiological forms, transthyretin-related hereditary amyloidosis (ATTRm), can present with several phenotypes, depending mainly on the specific mutation. We aimed to characterize the phenotype of patients with ATTRm due to Ile68Leu mutation, comparing them to patients with wild-type transthyretin amyloidosis (ATTRwt). Methods and results Data of 67 Ile68Leu ATTRm patients from two Italian referral centres (Bologna and Florence) were retrospectively analysed and compared to those of 82 ATTRwt patients. Fifty-five unaffected mutation carriers were also analysed. Cumulative disease onset was 50% at age 71. A total of 56/67 (84%) patients had a predominantly cardiac phenotype at presentation with concentric increase in left ventricular wall thickness [median 17 mm], and normal or near normal left ventricular ejection fraction (79% of patients). Low QRS voltages were present only in 29% of patients but voltage/mass ratio was low (0.5). Carpal tunnel syndrome was noted in 43%. The overall phenotypic profile was similar to ATTRwt but Ile68Leu ATTRm patients typically presented younger (median 71 vs. 78 years) and were more likely to have (mild) symptomatic neurological involvement (19% vs. 2%). Male prevalence was 44% in unaffected mutation carriers and 78% in affected patients. Age-adjusted survival was comparable between groups. Conclusions Ile68Leu ATTRm is a cause of familial amyloidotic cardiomyopathy endemic in central-northern Italy and presents as hypertrophic/restrictive cardiomyopathy quite similar to ATTRwt. Male preponderance is present in affected patients but not in unaffected mutation carriers. Age-adjusted survival is similar to ATTRwt.

Background Transthyretin amyloidosis is a life-threatening disorder caused by the deposition of TTR amyloid in various tissues and organs. The most common worldwide pathogenic variant with almost exclusive cardiac involvement is Val122Ile (rs76992529), with an allele frequency of 3.5% in the U.S. African-American population, but rare in Caucasians. Unexpectedly, we identified 23 Caucasian individuals with Val122Ile in our amyloidosis referral center (9 affected patients, 14 carriers), belonging to 9 unrelated families. Purpose To determine the ancestral origin of the Tuscan founder population of TTR Val122Ile carriers. Methods A total of 24 individuals were included in the analysis (our 23 probands and relatives from Val122Ile families and the Caucasian reference sample NA10851 (CEU – Utah resident with European ancestry). All samples were genotyped using the EUROFORGEN Global AIM-SNP array1, inclusive of 127 highly informative SNPs to infer genetic ancestry. We have performed a principal component analysis (PCA) of the 9 unrelated probands and NA10851, compared with the Phase 3 of the 1000 Genomes Project data, comprising 2504 unrelated individuals from >20 distinct populations.(Figure 1). Results As shown in Figure 1, all our samples but one (from Argentina) cluster very close to the super-cluster of European populations, and distant from the populations of African ancestry. The proband from Argentina and the Caucasian reference sample NA10851 cluster close to Mexicans and Peruvians, and the super-cluster of European populations, respectively, confirming the robustness of the analysis. Conclusion Based on this result, we can confidently conclude that our samples from Tuscan families in which the TTR Val122Ile variant segregates are of ancestral European origin, with no mixed African ancestry, implying that the same variant originated in Africans and Europeans independently and not as result of genetic admixture. These findings suggest the presence of a mutational hot spots in TTR, with potential impact on the epidemiology of amyloidosis worldwide. Acknowledgement/Funding The present study was supported by an Investigator-Initiated Research to Azienda Ospedaliero Universitaria Careggi from Pfizer Srl.

Transthyretin (TTR) gene has a causal role in a hereditary form of amyloidosis (ATTRm) and is potentially involved in the risk of senile systemic amyloidosis (SSA). To understand the genetics of ATTRm and SSA, we conducted a phenome-wide association study of TTR gene in 361,194 participants of European descent testing coding and non-coding variants. Among the 382 clinically-relevant phenotypes tested, TTR non-coding variants were associated with 26 phenotypic traits after multiple testing correction. These included signs related to both ATTRm and SSA such as chronic ischaemic heart disease (rs140226130, p=2.00×10−6), heart failure (rs73956431, p=2.74×10−6), atrial fibrillation (rs10163755, p=4.63×10−6), dysphagia (rs2949506, p=3.95×10−6), intestine diseases (rs970866, p=7.14×10−6) and anxiety (rs554521234, p=8.85×10−6). Consistent results were observed for TTR disease-causing mutation Val122Ile (rs76992529) with respect to carpal tunnel syndrome (p=6.41×10−6) and mononeuropathies of upper limbs (p=1.22×10−5). Sex differences were also observed in line with ATTRm and SSA epidemiology. Additionally, we explored possible modifier genes related to TTR function, observing convergent associations of RBP4 variants with the clinical phenotypes associated with TTR locus. In conclusion, we provide novel insights regarding the molecular basis of ATTRm and SSA based on large-scale cohort, expanding our understanding of the phenotypic spectrum associated with TTR gene variation.

Either 99mTechnetium diphosphonate (Tc-DPD) or pyrophosphate (Tc-PYP) scintigraphy plays a relevant role in diagnosing transthyretin cardiac amyloidosis (CA), and labeled radiotracers have been extensively studied in diagnosing CA. Few studies have analyzed and validated 99mTc-Hydroxymethylene diphosphonate (Tc-HMDP). Our aim was to validate the diagnostic accuracy of Tc-HMDP total-body scintigraphy in a cohort of patients with biopsy-proven transthyretin CA.We retrospectively evaluated all patients undergoing 99mTc-HMDP total-body scintigraphy, in adjunct to a comprehensive diagnostic work-up for suspected CA. Sixty-five patients were finally diagnosed with CA, while it was excluded in 20 subjects with left ventricular hypertrophy of various etiologies. Twenty-six patients had AL-CA, 39 had TTR CA (16 TTRm, 23 TTRwt). At Tc-HMDP scintigraphy, 2 AL patients showed a Perugini score grade 1 heart uptake, while 24 showed no uptake. All TTR patients showed Tc-HMDP uptake, with three patients showing a Perugini score grade 1, 16 grade 2, and 20 grade 3, respectively. No uptake was observed in patients with left ventricular hypertrophy. A positive Tc-HMDP scintigraphy showed a 100% sensitivity and a 96% specificity for TTR CA identification.Tc-HMDP scintigraphy is as accurate as Tc-DPD or Tc-PYP, and may therefore de facto be considered a valuable tool for the diagnosis of TTR CA.

Aim Several studies suggest that the N-terminal fragment of pro-brain natriuretic peptide levels are quite different in wild-type transthyretin (TTR)-related amyloidosis (ATTRwt) and mutated TTR-related amyloidosis (ATTRm) compared with immunoglobulin light-chain cardiac amyloidosis. Our aim was to test this hypothesis in a cohort of patients with different types of cardiac amyloidosis. Patients and methods Seventy patients with ATTRwt, ATTRm, and light-chain cardiac amyloidosis matched for left ventricular (LV) mass index were studied by standard echocardiography, tissue Doppler imaging, and plasmatic cardiac biomarkers. Results Despite similar LV mass and renal function, patients with ATTR cardiac amyloidosis showed lower levels of N-terminal fragment of pro-brain natriuretic peptide than do light-chain amyloidosis ones, especially when expressed as a function of LV mass index. Conclusion Amyloidogenic light-chain-derived fibrils induce more severe myocardial dysfunction in light-chain amyloidosis than in ATTR, despite similar myocardial infiltration. Thus, the degree of cardiac dysfunction may be related not only to the amount of amyloid deposited, but also to qualitative differences among fibrils.

The aim of this study was to validate noninvasive prenatal testing (NIPT) for fetal aneuploidies by whole-genome massively parallel sequencing (MPS).MPS was performed on cell-free DNA (cfDNA) isolated from maternal plasma in two groups: a first set of 186 euploid samples and a second set of 195 samples enriched of aneuploid cases (n = 69); digital PCR for fetal fraction (FF) assessment was performed on 178/381 samples. Cases with 10 × 10All positive samples for trisomy 21 (T21) (n = 43), trisomy 18 (T18) (n = 6) and trisomy 13 (T13) (n = 7) were correctly identified (sensitivity: 99.9%); 5 false positive results were reported: 3 for T21 (specificity = 98.9%) and 2 for T13 (specificity = 99.4%). Besides FF, total cfDNA concentration seems another important parameter for MPS, since it influences the number of reads.The overall test accuracy allowed us introducing NIPT for T21, T18 and T13 as a clinical service for pregnant women after 10 + 4 weeks of gestation. Sex chromosome aneuploidy assessment needs further validation due to the limited number of aneuploid cases in this study.

Transthyterin amyloidosis is a life-threatening disorder caused by the deposition of hepatocyte-derived transthyretin (TTR) amyloid in various tissues and organs. The most common worldwide pathogenic variant with almost exclusive cardiac involvement is Val142Ile with an allele frequency of 3.5% in U.S. African-American population, but supposed extremely rare, with only sporadic cases in Caucasian patients. Unexpectedly, in our amyloidosis referral centre, we identified five patients (15.1% of all TTRm diagnosed patients, three families, two singleton) with Val142Ile variant belonging to unrelated families of Caucasian origin. Molecular study was performed in a total of 10 individuals of which three were Italian families (three affected individuals and five unaffected individuals) and two were singleton (one Italian patient and one patient from Argentine with Spanish ancestry). Sequence analysis of TTR gene revealed the presence of the heterozygous Val142Ile in the five affected patients and in five asymptomatic individuals. All probands underwent, at diagnosis, a complete clinical, echocardiographic and biohumoral evaluation. To the best of our knowledge, we describe the larger report of Caucasian patients with Val142Ile cardiomyopathy. All patients at diagnosis showed symptoms of heart failure with increased thickness of left ventricular walls and systo-diastolic left ventricular dysfunction. They also showed increased plasma values of NT-proBNP and troponin I. Our data confirm that Caucasian patients with the Val142Ile pathogenic variant have phenotypic manifestations similar to that of African-American one. Moreover, our data clearly show that Val142Ile pathogenic variant is not only an African-American mutation but could be also an underestimated Caucasian variant.

BACKGROUND Optimization of factor VIII (FVIII) infusion in hemophilia A would benefit from identification of FVIII pharmacokinetics (PK) determinants. The low-density lipoprotein receptor (LDLR) contains an FVIII-binding site and might influence FVIII clearance. Consistently, LDLR polymorphisms have been associated with FVIII levels. OBJECTIVE To investigate the relationships between individual FVIII PK and functional LDLR polymorphisms. PATIENTS/METHODS Thirty-three hemophilia A patients (FVIII coagulant activity [FVIII:C] ≤2 IU/dL) without inhibitors underwent 85 FVIII single-dose (21.4-51.8 IU/kg) PKs with different FVIII concentrates. Twenty patients underwent repeated PKs (2-6). FVIII C measured up to 72 hours was analyzed by two-compartment model. Parameters were evaluated in relation to F8 mutations, ABO blood-group and LDLR genotypes. RESULTS F8 mutation types were not associated with PK parameters. ABO and LDLR c.1773C/T polymorphism were associated with Alpha, Alpha HL, CLD2, K1-2, and K2-1 parameters, suggesting an influence on the FVIII initial distribution phase. Regression analysis showed an independent association of both ABO and LDLR c.1773C/T with PK parameters (Alpha, β-coefficient -0.311 vs 0.348; CLD2, β-coefficient -0.335 vs 0.318), giving rise to an additive effect in subjects stratified by combined phenotypes. Differently, the LDLR c.81C/T was associated with FVIII clearance and volume of distribution at steady state, which could be related to distinct effects of polymorphisms, potentially linked to LDLR intracellular distribution and FVIII binding behavior. CONCLUSIONS With the limitation of different FVIII concentrates and low number of patients, our data show plausible associations of LDLR polymorphisms with FVIII PK parameters, thus supporting their investigation as candidate functional determinants of FVIII PK.

Background and purpose: Cerebrovascular complications are often the first cause of hospitalization in patients with Fabry disease (FD). Screenings for FD among stroke patients have yielded discrepant results, likely as a result of heterogeneous or incomplete assessment. We designed a study to identify FD among adults 60 years of age or younger who were consecutively admitted for acute ischemic stroke or transient ischemic attack (TIA) to a stroke neurology service in Italy. Methods: Patients with first-ever or recurrent events were included, irrespective of gender, risk factors, or stroke type. We screened male patients using α-galactosidase A enzyme assay, and female patients using DNA sequencing. FD was eventually established after a broad multidisciplinary discussion. Results: We screened 108 patients (61% males, median age: 48 years); 84% of these patients had stroke. De novo FD diagnosis was established in 3 patients (2.8%; 95% confidence interval, .57-8.18): a 59-year-old man with recurrent lacunar-like strokes and multiple risk factors; a 42-year-old woman with recurrent cryptogenic minor strokes; and a 32-year-old woman with recurrent strokes previously attributed to Behcet's disease. Screened patients were systematically asked for typical FD symptoms; each of the de novo patients reported one or more of the following: episodes of hand/foot pain during fever, angiokeratoma, and family history of heart disease. In all of the patients events were recurrent, and lacunar-like infarcts characterized their brain imaging. Conclusions: Prevalence of FD among nonselected adults 60 years of age or younger with acute ischemic stroke or TIA is not negligible. A systematic search for FD in a stroke setting, using a comprehensive clinical, biochemical, and genetic screening protocol, may be worthwhile.

Background Full body scintigraphy using bone tracers plays an important role in defining the type of amyloidosis and in diagnosing the heart involvement (cardiac amyloidosis, CA). No study has been conducted to explore lung retention (LR) in CA and its correlation to heart retention (HR).We evaluated LR in patients undergoing 99mTc-HMDP scintigraphy during evaluation for suspected CA. Methods and results We enrolled 93 suspected CA patients. Patients underwent a complete diagnostic work up. After diagnostic process 82 patients resulted affected by certain CA (20 AL and 62 TTR), while 11 subjects showed left ventricular hypertrophy (LVH) not caused by CA. 99mTc-HMDP cardiac uptake was evaluated using the Perugini visual score while the modified Janssen score was used for LR estimation (grade 0 no uptake, grade 1 less than ribs, grade 2 more than ribs). Results 99mTc-HMDP LR was observed in 1/20 AL patient (5%), while 36/62 (58%) TTR patients showed LR with 29 grade 1 (47%) and 7 grade 2 (11%). No LR was observed in patients with LVH and no CA. LR was not evident in patients without HR, present in 1/3 (33%) of the patients with Perugini 1 HR and 11/24 (46%) and 26/36 (72%) of the patients showing respectively a Perugini 2 and a Perugini 3. Conclusion 99mTc-HMDP scintigraphy shows LR in about 60% of TTR subjects, related to the grade of HR. In AL amyloidosis LR is less frequent than in TTR amyloidosis suggesting an aetiological tropism that seems comparable to the already known TTR related cardiac tropism.

Next generation sequencing (NGS) represents the newest approach for massive DNA sequencing. Although this innovative approach is a well-established point in diagnostic genetics, the applicability in forensic field is still limited to sporadic pilot studies. This limit arises from the samples' complexity, in terms of poor quality/quantity ratio, leading to a difficult setting in analytic systems. In this study, first NGS forensic data with a new analytic protocol are presented; data were obtained with a pre-release version of the MiSeq system (Illumina). The system is able to sequence, at the same time, a wide panel including autosomal 63 STRs and 95 SNPs commonly used in forensic practice. In the first part of the study sensitivity and concordance studies were performed by using DNA controls with known genetic profile; also mixtures with different male/female concentrations were analysed, in order to verify the panel performance in comparison with traditional kits. In the second part of the experiment, 60 casework samples coming from different tissues, biological fluids, traces on supports, lysates, different aged/degraded samples were analysed. Bioinformatic analysis was performed with MiSeq FGx System BETA version (Illumina), an innovative software with an easy interface that permits a rapid data interpretation by filtering the big data quantity generated by the instrument. The comparison between new data and genetic profiles obtained with traditional typing on the same samples, the sensitivity and the potential of NGS approach represents an interesting starting point in introducing this innovative approach on forensic genetics scenario.

This paper has been commented on by Marc Mugmon. Please see his response here . (Published: 1 April 2015) Citation: Journal of Community Hospital Internal Medicine Perspectives 2015, 5 : 26931 - http://dx.doi.org/10.3402/jchimp.v5.26931

We describe a novel transthyretin mutation in which phenylalanine is replaced with isoleucine in exon 3 at codon 64: Phe64Ile. The mutation was found in an isolated patient and it was not possible to perform a family study. The phenotype included heart and peripheral nerve involvement associated with a possible gastrointestinal and renal involvement.

The prevalence and clinical profile of cardiac troponin T gene mutations were evaluated in 150 consecutive patients with hypertrophic cardiomyopathy from the well-defined geographic region of Tuscany. Troponin T mutations had a low prevalence (3.3%; including a newly described Phe110Leu mutation) and were associated with heterogeneous clinical expression and outcome.