Rare exon 10 deletion in POLH gene in a family with xeroderma pigmentosum variant correlating with protein expression by immunohistochemistry.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease characterized by severe cutaneous and ocular sensitivity to sunlight, leading to skin cancer. Most XP patients belong to the XP complementation groups (XP-A to XP-G), due to mutations in genes involved in nucleotide excision repair (NER). On the other hand, the XP Variant type (XP-V, OMIM#278750), which accounts for about 20% of all XP patients, is associated with normal NER function. The disease gene is POLH, which encodes polymerase η (pol η) allowing translesion synthesis in regions of DNA damage. We observed an Italian family presenting with photosensitivity, freckling since childhood and multiple skin cancers. Complete sequence analysis of XPA, XPC, XPD/ERCC2 genes and exons 1-9 and 11 of POLH gene did not reveal pathological mutations. No PCR product was observed for exon 10 in POLH gene. By RT-PCR analysis followed by POLH exon 10 sequencing, all affected members were found to harbor a homozygous 170-nucleotide deletion. The same deletion was previously described in 3 XP-V families, one of southern Italian descent and two from Algeria, suggesting a possible founder mutation. The deletion determines a severe protein truncation and defective pol η activity. Immunohistochemical study showed markedly reduced pol η expression in skin lesions of the affected siblings compared to the normal control skin.

Genetic counselling and high-penetrance susceptibility gene analysis reveal the novel CDKN2A p.D84V (c.251A>T) mutation in melanoma-prone families from Italy.

Genetic susceptibility to primary cutaneous melanoma (PCM) may account for up to 12% of PCMs, presenting as the familial atypical mole/multiple melanoma syndrome (FAMMM), an autosomal dominant condition with incomplete penetrance and variable expressivity, characterized by PCM in at least two relatives and/or more than one PCMs in the same patient. To identify individuals at high genetic risk of PCM, from 1 January 2012 to 31 December 2015, we offered genetic counselling and molecular analysis of the two high-penetrance FAMMM susceptibility genes, cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase 4 (CDK4), to 92 consecutive, unrelated patients with FAMMM. Age at diagnosis and number of PCMs were obtained from medical records; the number of PCMs and affected relatives were recorded for each family. The diagnostic work-up consisted of genetic counselling and cascade genetic testing in patients and further extension to relatives of those identified as mutation carriers. All exons and exon/intron boundaries of CDKN2A and CDK4 genes were screened by direct bidirectional sequencing. We identified CDKN2A mutations in 19 of the 92 unrelated patients (20.6%) and in 14 additional, clinically healthy relatives. Eleven of these latter subsequently underwent excision of dysplastic nevi, but none developed PCM during a median follow-up of 37.3 months. In three patients from unrelated families, the novel CDKN2A p.D84V (c.251A>T) mutation was observed, associated with PCM in each pedigree. Genetic screening of FAMMM patients and their relatives can contribute towards specific primary and secondary prevention programmes for individuals at high genetic risk of PCM. The novel CDKN2A p.D84V (c.251A>T) mutation adds to the known mutations associated with FAMMM.

Incomplete penetrance of GLMN gene c.395-1G>C mutation in a family with glomuvenous malformations.

Glomuvenous malformations (GVMs, OMIM 138000) are hamartomas presenting in childhood as multiple, bluish, soft papules and nodules that tend to grow slowly in size and number with age. They are caused by autosomal dominant mutations in glomulin (GLMN) gene; penetrance varies from 80% at 20 to about 100% at age 30 years. We report on the c.395-1G>C mutation of GLMN gene in two siblings showing variable penetrance.

Glomuvenous malformations with smooth muscle and eccrine glands: unusual histopathologic features in a familial setting.

Glomuvenous malformations (OMIM 138000) are hamartomas presenting in childhood as multiple, bluish papules and nodules in the skin, which are characterized histopathologically by irregular vascular spaces surrounded by typical glomus cells. Glomuvenous malformations are caused by autosomal dominant mutations of the GLMN gene. A 34-year-old woman and her 16-year-old son presented with bluish papules and nodules since childhood. Biopsy specimens from both patients showed histopathologic features of glomuvenous malformations, unusually in consistent and close association with smooth muscle, hair follicles and eccrine glands. Sequencing of the GLMN gene revealed the p.C36X (c.108C>A) mutation in germline DNA from both patients. This is probably the first report describing the hamartomatous features of familial glomuvenous malformations consistently associated with a prominent smooth muscle component and eccrine glands.

Autosomal recessive atrial dilated cardiomyopathy with standstill evolution associated with mutation of Natriuretic Peptide Precursor A.

BACKGROUND: Atrial dilatation and atrial standstill are etiologically heterogeneous phenotypes with poorly defined nosology. In 1983, we described 8-years follow-up of atrial dilatation with standstill evolution in 8 patients from 3 families. We later identified 5 additional patients with identical phenotypes: 1 member of the largest original family and 4 unrelated to the 3 original families. All families are from the same geographic area in Northeast Italy. METHODS AND RESULTS: We followed up the 13 patients for up to 37 years, extended the clinical investigation and monitoring to living relatives, and investigated the genetic basis of the disease. The disease was characterized by: (1) clinical onset in adulthood; (2) biatrial dilatation up to giant size; (3) early supraventricular arrhythmias with progressive loss of atrial electric activity to atrial standstill; (4) thromboembolic complications; and (5) stable, normal left ventricular function and New York Heart Association functional class during the long-term course of the disease. By linkage analysis, we mapped a locus at 1p36.22 containing the Natriuretic Peptide Precursor A gene. By sequencing Natriuretic Peptide Precursor A, we identified a homozygous missense mutation (p.Arg150Gln) in all living affected individuals of the 6 families. All patients showed low serum levels of atrial natriuretic peptide. Heterozygous mutation carriers were healthy and demonstrated normal levels of atrial natriuretic peptide. CONCLUSIONS: Autosomal recessive atrial dilated cardiomyopathy is a rare disease associated with homozygous mutation of the Natriuretic Peptide Precursor A gene and characterized by extreme atrial dilatation with standstill evolution, thromboembolic risk, preserved left ventricular function, and severely decreased levels of atrial natriuretic peptide.

A novel mutation of the glomulin gene in an Italian family with autosomal dominant cutaneous glomuvenous malformations.

Glomuvenous malformations (GVM) are hamartomas characterized histologically by glomus cells, which should be distinguished from glomus tumors. Familial GVM are rare, often present as multiple lesions, and exhibit familial aggregation, with autosomal dominant transmission. GVM are caused by mutations of the glomulin (GLMN) gene on chromosome 1p21-p22. Their development is thought to follow the 'two-hit' hypothesis, with a somatic mutation required in addition to the inherited germline mutation. We describe a novel GLMN mutation in an Italian family with GVM in which some members present with the less commonly observed phenotype of solitary lesions. A second somatic 'hit' mutation in GLMN was not discovered in our family. We further provide histological, immunohistochemical and electron microscopic data exhibiting the classic features of GVM. The diagnosis of GVM is critical because of distinction from venous malformations and blue rubber bleb nevus syndrome, which may demonstrate clinical similarities but require different treatment.

Diagnostic work-up and risk stratification in X-linked dilated cardiomyopathies caused by dystrophin defects.

OBJECTIVES: We sought to describe the diagnostic work-up, phenotype, and long-term evolution of dilated cardiomyopathy (DCM) associated with Dystrophin (DYS) defects. BACKGROUND: X-linked DCM associated with DYS defects can be clinically indistinguishable from other types of DCM. METHODS: The series comprises 436 consecutive male patients diagnosed with DCM. Patients underwent endomyocardial biopsy (EMB). Genetic testing employed multiplex polymerase chain reaction and multiple ligation dependent probe assay for deletions and direct sequencing of the 79 exons and flanking regions of the gene for point mutations or small rearrangements. RESULTS: We identified DYS defects in 34 of 436 patients (7.8%) (onset age 34 ± 11 years, age range 17 to 54 years); 30 had proven X-linked inheritance. The 2 phenotypes included DCM with mild skeletal myopathy and/or increased serum creatine phosphokinase (n = 28) or DCM only (n = 6). The EMB showed defective dystrophin immunostain. The DYS defects consisted of 21 in-frame deletions and 11 out-of-frame deletions as well as 1 stop and 1 splice-site mutation. During a median follow-up of 60 months (interquartile range: 11.25 to 101.34 months) we observed 17 events, all related to heart failure (HF) (median event-free survival: 83.5 months). Eight patients (23%) underwent transplantation, and 9 (26%) died of HF while waiting for transplantation. Eight patients received an implantable cardioverter-defibrillator, although none had device intervention during a median follow-up of 14 months (interquartile range: 5 to 25 months). No patient died suddenly, suffered syncope, or developed life-threatening ventricular arrhythmias. CONCLUSIONS: DYS-related DCM should be suspected in male patients with increased serum creatine phosphokinase (82%) and X-linked inheritance. The disease shows a high risk of end-stage HF but a lower risk of life-threatening arrhythmias.

Mitochondrial cardiomyopathies: how to identify candidate pathogenic mutations by mitochondrial DNA sequencing, MITOMASTER and phylogeny.

Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause cardiomyopathy and heart failure. Owing to a high mutation rate, mtDNA defects may occur at any nucleotide in its 16 569 bp sequence. Complete mtDNA sequencing may detect pathogenic mutations, which can be difficult to interpret because of normal ethnic/geographic-associated haplogroup variation. Our goal is to show how to identify candidate mtDNA mutations by sorting out polymorphisms using readily available online tools. The purpose of this approach is to help investigators in prioritizing mtDNA variants for functional analysis to establish pathogenicity. We analyzed complete mtDNA sequences from 29 Italian patients with mitochondrial cardiomyopathy or suspected disease. Using MITOMASTER and PhyloTree, we characterized 593 substitution variants by haplogroup and allele frequencies to identify all novel, non-haplogroup-associated variants. MITOMASTER permitted determination of each variant's location, amino acid change and evolutionary conservation. We found that 98% of variants were common or rare, haplogroup-associated variants, and thus unlikely to be primary cause in 80% of cases. Six variants were novel, non-haplogroup variants and thus possible contributors to disease etiology. Two with the greatest pathogenic potential were heteroplasmic, nonsynonymous variants: m.15132T>C in MT-CYB for a patient with hypertrophic dilated cardiomyopathy and m.6570G>T in MT-CO1 for a patient with myopathy. In summary, we have used our automated information system, MITOMASTER, to make a preliminary distinction between normal mtDNA variation and pathogenic mutations in patient samples; this fast and easy approach allowed us to select the variants for traditional analysis to establish pathogenicity.

When should cardiologists suspect Anderson-Fabry disease?

Anderson-Fabry disease is a lysosomal storage disorder caused by α-galactosidase defects and progressive intracellular accumulation of globotriaosylceramide. The disease can be specifically treated with enzyme replacement therapy. Hemizygous men and heterozygous women can develop cardiac disease. Whereas men experience the most severe clinical phenotype, clinical presentation in women varies from asymptomatic to severely symptomatic. The characteristic cardiac phenotype is left ventricular hypertrophy mimicking sarcomeric hypertrophic cardiomyopathy or hypertensive heart disease. Early or prehypertrophy cardiac involvement may escape detection, unless electrocardiographic clues are present. The cardiac markers that raise suspicion of Anderson-Fabry disease include a short PR interval without a δ wave and a prolonged QRS interval, supraventricular and ventricular arrhythmias, and concentric left ventricular hypertrophy. Extracardiac features include renal failure, corneal deposits, and nervous, gastrointestinal, and cutaneous manifestations. Useful family data include cardiac and extracardiac traits in relatives and absence of male-to-male transmission. Symptoms are subtle, and the interval between the onset of symptoms and diagnosis may be as long as 20 years. As such, the diagnosis is typically late. Endomyocardial biopsy shows optically empty myocytes on light microscopy and dense osmiophilic bodies constituted of globotriaosylceramide on electron microscopy. Alpha-galactosidase A activity is reduced in hemizygous men but not in heterozygous women. Genetic testing is the gold standard for the diagnosis. In conclusion, a correct and timely diagnosis offers the possibility of disease-specific treatment that leads to sustained clinical benefits for cardiac and noncardiac signs and symptoms.

Mitochondrial DNA variant discovery and evaluation in human Cardiomyopathies through next-generation sequencing.

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We "shotgun" sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300x average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20x while heteroplasmic variants required >200x coverage. Several Sanger "misses" were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.

A combined histologic and molecular approach identifies three groups of gastric cancer with different prognosis.

The limited prognostic value of currently used histologic classifications of gastric cancer and their failure to account for the complexity of the disease as revealed by more recent investigations prompted a combined reinvestigation of histologic, molecular, and clinicopathologic patterns in 294 extensively sampled, invasive gastric cancers representing all main histotypes and stages of the disease and followed for a median of 150 months. Among histologic parameters tested, only cellular atypia, angio-lympho- or neuroinvasion, Ki67 proliferation index, expansile/infiltrative type growth, and T8 cell-rich high lymphoid intra-/peritumor response (HLR) proved to be stage-independent predictors of patient survival. Among molecular tests, p53 gene exon 7 (loop 3) and 8 (loop-sheet-helix motif and S-10 band), but not p53 protein overexpression, TP53 LOH or 18qLOH, were found to worsen prognosis. Microsatellite DNA instability was a favorable prognostic factor when coupled with HLR. Patient survival analysis of the main histotypes and their subtypes confirmed the favorable prognosis of HLR, well-differentiated tubular, muconodular, and low grade diffuse desmoplastic cancers, and highlighted the worse prognosis of anaplastic and infiltrative-lymphoinvasive mucinous cancers compared to ordinary cohesive and diffuse cancers. Distinct roles of individual morphologic and molecular factors in tumor progression of the different histotypes have been recognized. The combination of survival-predictive histotypes and individual histologic or molecular parameters allowed us to develop a classification of all gastric cancers into three grades of increasing malignancy which proved to be of high prognostic value.

The shortness of Pygmies is associated with severe under-expression of the growth hormone receptor.

The stature is a highly heritable trait controlled by genetic and environmental factors. The African Pygmies represent a paradigmatic example of non-disease-related idiopathic short stature (ISS), showing a similar endocrine profile of Caucasian individuals with ISS. Pygmy children show normal anthropometric and endocrine parameters until puberty, while adult Pygmies show normal baseline and post-stimulation serum growth hormone (GH) levels but low values of baseline serum GH-binding protein (GHBP) and insulin-like growth factor-I (IGF-I). This discrepancy suggests a defective response to GH occurring in adulthood since Pygmies lack both the pubertal serum IGF-I surge and the growth spurt. However, sequencing of the key genes of the GH-IGF-I axis failed to identify Pygmy specific variants or haplotypes. We therefore aimed at assessing whether the quantitative gene expression profile of two key genes of the GH-IGF-I axis, GH and GHR, was also similar in low-stature and normal stature populations. We showed that the GH gene expression is 1.8-fold reduced and the GH receptor (GHR) gene expression is 8-fold reduced in adult Pygmies in comparison with sympatric adult Bantu, and that this reduction is not associated with sequence variants of the GHR gene. The marked decrease of the GHR expression in Pygmies is associated with reduced serum levels of the IGF-I and GHBP. Our results, documenting a markedly reduced GHR gene expression in adult Pygmies, could contribute to elucidate the mechanisms involved in ISS in Caucasoid subjects.

Transcriptomic and proteomic analysis in the cardiovascular setting: unravelling the disease?

Whole gene expression analysis through microarray technologies revolutionized the manner of identifying changes in biological events and complex diseases, such as cardiovascular settings. These new methodologies may scan up to 35 000 transcripts at once rather than screening a small amount of genes one at a time. The ability of microarrays to provide a broad insight into the disease process directly within the tissues provides a unique insight into the intracellular perturbations of the cell organization and function and sheds an entirely unique new perspective on the heart failure process. Commonalities and differences at the molecular level will identify critical pathways of pathogenesis, and response to therapy, or both: indeed, gene expression profiling holds tremendous promise for classifying clinical phenotypes, developing prognostic predictors and, most importantly, providing novel unbiased insights into the mechanisms underlying heart disease and, eventually, novel causative genes. On the contrary, established proteomic technologies, together with the new alternative strategies currently under evaluation (i.e. metabolomics), are now making possible the translation of data obtained on the bench to the daily clinical routine with the discovery of new diagnostic/prognostic biomarkers (such as troponin for ACS and BNP for congestive heart failure) and the identification of new therapeutic approaches for combating heart diseases. Finally, genomic studies (including transcriptomics) together with proteomics should not represent a challenge for who is going to win the final battle, but rather they should provide a setting in which together and in a complementary fashion the final fight against heart disease can be won.

Barth syndrome associated with compound hemizygosity and heterozygosity of the TAZ and LDB3 genes.

Barth syndrome is an X-linked recessive disorder caused by the tafazzin (TAZ) gene mutations and includes dilated cardiomyopathy (DCM) with left ventricular non-compaction, neutropenia, skeletal myopathy, abnormal mitochondria and 3-methylglutaconic aciduria. Dilated cardiomyopathy with left ventricular non-compaction transmitted as an autosomal dominant condition has also been associated with LIM domain-binding 3 (LDB3) gene defects. We describe a family in which the 12-year-old proband had left ventricular non-compaction and DCM. His mother had five miscarriages, two other sons who died in infancy, and a healthy son and daughter. The proband showed left ventricular non-compaction-DCM, skeletal myopathy, recurrent oral aphthous ulcers and cyclic neutropenia. The DCM progressively improved with age; medical therapy was discontinued at 5 years of age. At present, left ventricular function is normal and arrhythmias are absent. Magnetic resonance imaging documented left ventricular non-compaction. However, oral aphthous ulcers and cyclic neutropenia have recurred. In the proband we identified two novel mutations, one of maternal origin in the TAZ gene (p.[Glu202ValfsX15]) and one of paternal origin in the LDB3 gene (p.[Thr350Ile]). The mother, brother and father are healthy; although the latter two show prominent left ventricle trabeculation without dysfunction. Expression studies of TAZ and LDB3 genes were conducted in family members and controls. In the proband, brother and father, LDB3 expression was similar to control cases. TAZ and LDB3 expression progressively declined with age in control both blood and myocardial samples. However, an endomyocardial biopsy performed in the proband at 6 months of age, showed significantly lower TAZ and LDB3 expression than in age-matched myocardial controls. We believe that the clinical, genetic and expression data support the hypothesis that tafazzins are essential during fetal and early post-natal life.

Two novel and one known mutation of the TGFBR2 gene in Marfan syndrome not associated with FBN1 gene defects.

TGF-beta-receptor 2 (TGFBR2) gene defects have been recently associated with Marfan syndrome (MFS) with prominent cardio-skeletal phenotype in patients with negative fibrillin-1 (FBN1) gene screening. Four mutations have been identified to date in five unrelated families. We screened TGFBR2 gene by direct automated sequencing in two adult patients diagnosed with MFS according to Ghent criteria, and in one girl clinically suspected as affected on the basis of a major cardiovascular criterion and skeletal involvement, all proven not to carry mutations in the exon-intron boundaries of FBN1 gene. We identified two novel and one known TGFBR2 gene mutations in the three unrelated probands. The D446N was identified in a 4-year-old girl with de novo disease characterized by severe cardiovascular disease and skeletal involvement. The M425V and R460H mutations were identified in two familial, autosomal dominant MFSs, both characterized by major cardio-skeletal signs and absence of major ocular signs. The mutation R460H has been recently reported in a family with thoracic aortic aneurysms and dissection. The three mutations are absent in 192 controls and affect evolutionarily conserved residues of the serine/threonine kinase domain (exon 5). Our data support the recently reported association between TGFBR2 gene and MFS without major ocular signs (MFS2). The number of genotyped cases however is too low to confirm that major ocular signs are characteristically absent in MFS2. Accordingly, all patients proven or suspected to be affected by MFS with negative FBN1 gene screening could benefit from rapid investigation of the TGFBR2 gene.

A novel AbetaPP mutation exclusively associated with cerebral amyloid angiopathy.

Mutations in AbetaPP cause deposition of Abeta amyloid fibrils in brain parenchyma and cerebral vessels, resulting in Alzheimer's disease (AD) and/or cerebral amyloid angiopathy (CAA). We report a novel mutation (L705V) within the Abeta sequence of AbetaPP in a family with autosomal dominant, recurrent intracerebral hemorrhages. Pathological examination disclosed severe CAA, without parenchymal amyloid plaques or neurofibrillary tangles. This variant highlights the vascular tropism of mutated Abeta, resulting in CAA instead of the pathological hallmarks of AD.

A novel mtDNA point mutation in tRNA(Val) is associated with hypertrophic cardiomyopathy and MELAS.

BACKGROUND: Pathological mutations of mitochondrial (mt) DNA may cause specific diseases such as cardiomyopathies or hearing loss, or syndromes such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome. We describe a novel mtDNA mutation in a patient with severe hypertrophic cardiomyopathy associated with MELAS. The familial phenotype included 1) hypertrophic cardiomyopathy and MELAS, 2) clinically mild cardiac hypertrophy, and 3) deafness. METHODS: The proband and her first degree relatives underwent echo and electrocardiograms, and biochemical tests. Magnetic resonance imaging of the brain was performed in the proband. mtDNA was fully analyzed by sequencing. DNA purification, polymerase chain reaction and direct automated sequencing were performed following standard procedures. Heteroplasmy of the novel mutation was quantified by densitometric analysis. RESULTS: A novel G1644A transition affecting the tRNA(Val) was identified in the proband and maternal relatives. The mutation has been interpreted as pathological because the G at the 1644 position is a highly conserved base, is heteroplasmic with higher levels of mutant DNA in the proband than in the relatives, is located in the unique tRNA(Val), is very close to a mutation described as causative of MELAS, and finally has not been found in 100 healthy controls. CONCLUSIONS: Although it is rare for patients with MELAS to be referred to cardiological evaluation because of coexisting cardiomyopathy, cardiologists should be aware of this association as well as of the non cardiac signs that may address the diagnosis to mtDNA defect-related disease in families with a variable phenotype.