Combined exome and whole transcriptome sequencing identifies a de novo intronic SRCAP variant causing DEHMBA syndrome with severe sleep disorder.

Rare heterozygous variants in exons 33-34 of the SRCAP gene are associated with Floating-Harbor syndrome and have a dominant-negative mechanism of action. At variance, heterozygous null alleles falling in other parts of the same gene cause developmental delay, hypotonia, musculoskeletal defects, and behavioral abnormalities (DEHMBA) syndrome. We report an 18-year-old man with DEHMBA syndrome and obstructive sleep apnea, who underwent exome sequencing (ES) and whole transcriptome sequencing (WTS) on peripheral blood. Trio analysis prioritized the de novo heterozygous c.5658+5 G > A variant. WTS promptly demostrated four different abnormal transcripts affecting >40% of the reads, three of which leading to a frameshift. This study demonstrated the efficacy of a combined ES-WTS approach in solving undiagnosed cases. We also speculated that sleep respiratory disorder may be an underdiagnosed complication of DEHMBA syndrome.

Specifications and validation of the ACMG/AMP criteria for clinical interpretation of sequence variants in collagen genes associated with joint hypermobility.

Deleterious variants in collagen genes are the most common cause of hereditary connective tissue disorders (HCTD). Adaptations of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria are still lacking. A multidisciplinary team was set up for developing specifications of the ACMG/AMP criteria for COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL11A1, COL11A2 and COL12A1, associated with various forms of HCTD featuring joint hypermobility, which is becoming one of the most common reasons of referral for molecular testing in this field. Such specifications were validated against 209 variants, and resulted effective for classifying as pathogenic and likely pathogenic null alleles without downgrading of the PVS1 level of strength and recurrent Glycine substitutions. Adaptations of selected criteria reduced uncertainties on private Glycine substitutions, intronic variants predicted to affect the splicing, and null alleles with a downgraded PVS1 level of strength. Segregation and multigene panel sequencing data mitigated uncertainties on non-Glycine substitutions by the attribution of one or more benignity criteria. These specifications may improve the clinical utility of molecular testing in HCTD by reducing the number of variants with neutral/conflicting interpretations. Close interactions between laboratory and clinicians are crucial to estimate the a priori utility of molecular test and to improve medical reports.

Compound Heterozygosity for OTOA Truncating Variant and Genomic Rearrangement Cause Autosomal Recessive Sensorineural Hearing Loss in an Italian Family.

Hearing loss (HL) affects 1-3 newborns per 1000 and, in industrialized countries, recognizes a genetic etiology in more than 80% of the congenital cases. Excluding GJB2 and GJB6, OTOA is one of the leading genes associated with autosomal recessive non-syndromic HL. Allelic heterogeneity linked to OTOA also includes genomic rearrangements facilitated by non-allelic homologous recombination with the neighboring OTOAP1 pseudogene. We present a couple of Italian siblings affected by moderate to severe sensorineural hearing loss (SNHL) due to compound heterozygosity at the OTOA locus. Multigene panel next-generation sequencing identified the c.2223G>A, p.(Trp741*) variant transmitted from the unaffected mother. Assuming the existence of a second paternal deleterious variant which evaded detection at sequencing, genomic array analysis found a ~150 Kb microdeletion of paternal origin and spanning part of OTOA. Both deleterious alleles were identified for the first time. This study demonstrates the utility of an integrated approach to solve complex cases and allow appropriate management to affected individuals and at-risk relatives.

Phenotypic Variability of a Pathogenic PKP2 Mutation in an Italian Family Affected by Arrhythmogenic Cardiomyopathy and Juvenile Sudden Death: Considerations From Molecular Autopsy to Sport Restriction.

Background: Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder with an estimated prevalence between 1:2,000 and 1:5,000 and is characterized by the fibrofatty replacement of cardiomyocytes that predisposes to malignant arrhythmias, heart failure, and sudden cardiac death. The diagnosis is based on the 2010 Task Force Criteria including family history, electrocardiographic traits and arrhythmogenic pattern, specific gene mutations, and structural and/or histological abnormalities. Most ACMs display an autosomal dominant mode of inheritance often with incomplete penetrance and variable expressivity. Genetic screening of patients with ACM identifies pathogenic or likely pathogenic variants, prevalently in genes encoding the cardiac desmosome (PKP2, DSP, DSC2, DSG2, and JUP) or less frequently in non-desmosomal genes (CTNNA3, PLN, TMEM43, RYR2, SCN5A, CDH2, and DES). Methods: In the present study, we performed molecular autopsy in a boy who died suddenly during physical exertion. In addition to post-mortem examination, a DNA sample was analyzed with next-generation sequencing (NGS). Results: The genetic analysis revealed the presence of pathogenic heterozygous c.314del (p.Pro105Leufs*7) frameshift variant in the PKP2 gene. Cascade screening of family members allowed us to identify 12 mutation carriers and to intervene on subjects at risk, many of whom were athletes. Conclusions: Molecular autopsy can establish cardiogenetic diagnosis and allow appropriate preventative measures in high-risk relatives.

Rare Somatic MEN1 Gene Pathogenic Variant in a Patient Affected by Atypical Parathyroid Adenoma.

OBJECTIVE: Atypical parathyroid adenoma is a rare neoplasm, showing atypical histological features intermediate between classic benign adenoma and the rarest parathyroid carcinoma, whose the clinical behaviour and outcome is not yet understood or predictable. Up to date only two cases of atypical adenoma were found associated to a MEN1 syndrome, and only one was proved to carry a pathogenic variant of the MEN1 gene. DESIGN: We report the clinical, histologic, and molecular findings of a 44-year-old woman, presenting with a histologically proved atypical parathyroid adenoma with an apparent aggressive behaviour. METHODS AND RESULTS: CDC73 gene was screened at germline and somatic levels with no results. Whole exome sequencing performed on DNA extracted from blood leukocytes and tumour tissue revealed a somatic MEN1 gene heterozygous variant, c.912+1G > A, of the splicing donor site of exon 6. On immunohistochemistry, downregulation of the menin protein expression in the neoplastic cells was also observed. CONCLUSIONS: We report the second case of a rare association of a somatic MEN1 gene mutation in a patient with atypical parathyroid adenoma. We suggest that MEN1 gene could be an underestimate genetic determinant of these rare histological entities, and we highlight the utility of a complete genetic screening protocol, by the use of next-generation sequencing technology in such undetermined clinical cases with no frank clinical presentation.

The recurrent SETBP1 c.2608G > A, p.(Gly870Ser) variant in a patient with Schinzel-Giedion syndrome: an illustrative case of the utility of whole exome sequencing in a critically ill neonate.

BACKGROUND: Schinzel-Giedion syndrome (SGS) is a multiple malformation syndrome mainly characterized by severe intellectual disability, distinctive facial features, and multiple congenital anomalies, including skeletal abnormalities, genitourinary and renal malformations, cardiac defects, as well as an increased pediatric cancer risk. Recently, SGS has been associated with de novo heterozygous deleterious variants in the SETBP1 gene; to date, nine different variants, clustering in exon 4 of SETBP1, have been identified in 25 patients. CASE PRESENTATION: In this study, by using Whole Exome Sequencing (WES), we identified a patient with a recurrent missense mutation in SETBP1, the c.2608G > A, p.(Gly870Ser) variant, previously reported as likely pathogenic. This finding allowed us to confirm the suspected clinical diagnosis of SGS. Clinical features of patients carrying the same variant, including our patient, were evaluated by a review of medical records. CONCLUSIONS: Our study confirms SGS as a severe disorder potentially presenting at birth as a critically ill neonate and demonstrates the causal role of the c.2608G > A, p.(Gly870Ser) variant in the etiology of the syndrome. Moreover, although the cohort of SETBP1-patients reported in the literature is still small, our study reports for the first time the prevalence of the variant (about 27%, 7/26). Finally, given the heterogeneity of clinical presentations of affected patients hospitalized in Neonatal Intensive Care Units (NICU) and/or Pediatric Intensive Care Units (PICU), in agreement with emerging data from the literature, we suggest that WES should be used in the diagnosis of unexplained syndromic conditions, and even as part of a standard first-line diagnostic approach, as it would allow a better diagnosis, counseling and management of affected patients and their families.

Compound Phenotype Due to Recessive Variants in LARP7 and OTOG Genes Disclosed by an Integrated Approach of SNP-Array and Whole Exome Sequencing.

Neurodevelopmental disorders are a challenge in medical genetics due to genetic heterogeneity and complex genotype-phenotype correlations. For this reason, the resolution of single cases not belonging to well-defined syndromes often requires an integrated approach of multiple whole-genome technologies. Such an approach has also unexpectedly revealed a complex molecular basis in an increasing number of patients, for whom the original suspect of a pleiotropic syndrome has been resolved as the summation effect of multiple genes. We describe a 10-year-old boy, the third son of first-cousin parents, with global developmental delay, facial dysmorphism, and bilateral deafness. SNP-array analysis revealed regions of homozygosity (ROHs) in multiple chromosome regions. Whole-exome sequencing prioritized on gene-mapping into the ROHs showed homozygosity for the likely pathogenic c.1097_1098delAG p. (Arg366Thrfs*2) frameshift substitution in LARP7 and the likely pathogenic c.5743C>T p.(Arg1915*) nonsense variant in OTOG. Recessive variants in LARP7 cause Alazami syndrome, while variants in OTOG cause an extremely rare autosomal recessive form of neurosensorial deafness. Previously unreported features were acrocyanosis and palmoplantar hyperhidrosis. This case highlights the utility of encouraging technological updates in medical genetics laboratories involved in the study of neurodevelopmental disorders and integrating laboratory outputs with the competencies of next-generation clinicians.

Double missense mutations in cardiac myosin-binding protein C and myopalladin genes: A case report with diffuse coronary disease, complete atrioventricular block, and progression to dilated cardiomyopathy.

Cardiomyopathies caused by double gene mutations are rare but conferred a remarkably increased risk of end-stage progression, arrhythmias, and poor outcome. Compound genetic mutations leading to complex phenotype in the setting of cardiomyopathies represent an important challenge in clinical practice, and genetic tests allow risk stratification and personalized clinical management of patients. We report a case of a 50-year-old woman with congestive heart failure characterized by dilated cardiomyopathy, diffuse coronary disease, complete atrioventricular block, and missense mutations in cardiac myosin-binding protein C (MYBPC3) and myopalladin (MYPN). We discuss the plausible role of genetic profile in phenotype determination.

Sudden death in mild hypertrophic cardiomyopathy with compound DSG2/DSC2/MYH6 mutations: Revisiting phenotype after genetic assessment in a master runner athlete.

Cardiomyopathies represent a well-known cause of heart failure and sudden death. Although cardiomyopathies are generally categorized in distinct nosographic entities, characterized by single gene-to-disease causal relationships, recently, oligogenic mutations have also been associated to relevant cardiac clinical features. We report the case of a master athlete carrying trigenic mutations in desmoglein-2 (DSG2), desmocollin-2 (DSC2) and heavy chain myosin 6 (MYH6), which determine a mild hypertrophic phenotype associated both to ventricular tachyarrhythmias and atrio-ventricular block. We discuss the differential diagnosis and prognostic approach in patient affected by complex cardiomyopathy phenotype, along with the importance of sport restriction and sudden death prevention.

Sudden cardiac death in J wave syndrome with short QT associated to a novel mutation in Nav 1.8 coding gene SCN10A: First case report for a possible pharmacogenomic role.

INTRODUCTION: Sudden cardiac death is an important cause of mortality in the general population. It represents an important challenge for clinicians, often being the only symptom of a broad spectrum of cardiac pathologies and inherited heart conditions. Early repolarization syndrome and Brugada syndrome are part of the wider "J-wave" syndrome, which may also include the short QT syndrome as a third factor of an ionic channel imbalance in the arrhythmogenic landscape. CASE PRESENTATION: We describe the case of a woman struck down by sudden cardiac death, with short QT and early repolarization, in which we found an extremely rare and putatively pathogenic heterozygous variant in the SCN10A gene. Variants involving SCN10A, which encodes a voltage-gated sodium channel, were already associated with alterations of cardiac conduction parameters and the cardiac rhythm disorder, thereby influencing the cardiac physiology and predisposing to arrhythmia. CONCLUSION: We underline the role of genetic predisposition to sudden cardiac death and, for the first time, suggest a possible environmental effect, such as a pharmacological therapy in the setting of sudden death, with the purpose to increase awareness in clinical practice.

A novel mutation in CDH11, encoding cadherin-11, cause Branchioskeletogenital (Elsahy-Waters) syndrome.

Cadherins are cell-adhesion molecules that control morphogenesis, cell migration, and cell shape changes during multiple developmental processes. Until now four distinct cadherins have been implicated in human Mendelian disorders, mainly featuring skin, retinal and hearing manifestations. Branchio-skeleto-genital (or Elsahy-Waters) syndrome (BSGS) is an ultra-rare condition featuring a characteristic face, premature loss of teeth, vertebral and genital anomalies, and intellectual disability. We have studied two sibs with BSGS originally described by Castori et al. in 2010. Exome sequencing led to the identification of a novel homozygous nonsense variant in the first exon of the cadherin-11 gene (CDH11), which results in a prematurely truncated form of the protein. Recessive variants in CDH11 have been recently demonstrated in two other sporadic patients and a pair of sisters affected by BSGS. Although the function of this cadherin (also termed Osteoblast-Cadherin) is not completely understood, its prevalent expression in osteoblastic cell lines and up-regulation during differentiation suggest a specific function in bone formation and development. This study identifies a novel loss-of-function variant in CDH11 as a cause of BSGS and supports the role of cadherin-11 as a key player in axial and craniofacial malformations.

Refinement of the critical 7p22.1 deletion region: Haploinsufficiency of ACTB is the cause of the 7p22.1 microdeletion-related developmental disorders.

Non-recurrent microdeletion (≤2 Mb in size) in 7p22.1 is a rarely described cytogenetic aberration, only recently reported in patients with developmental delay/intellectual disability, short stature and microcephaly. The size of the deletions ranged from 0.37 to 1.5 Mb, and reported genotype-phenotype correlations identified a minimum deleted region of 0.37 Mb involving the FBLX18, ACTB, FSCN1, RNF216 and ZNF815P genes. The authors suggested that deletion of ACTB, which encodes ß-actin, an essential component of the cytoskeleton, could be responsible for the clinical features observed in the patients with a 7p22.1 microdeletion. Here, we describe a 23-month-old child displaying developmental delay, short stature, microcephaly and distinctive facial features. Chromosomal microarray analysis performed using high-resolution SNP-array platform revealed a de novo interstitial 60 Kb microdeletion in the 7p22.1 region (from 5,509,127 bp to 5,569,096 bp, hg19) encompassing only two genes: FBXL18 and ACTB. To the best of our knowledge, this is the smallest deletion at 7p22.1 to date reported in medical literature (Pubmed). Combining our data with phenotypic and genotypic data of cases from literature, we were able to narrow the minimal critical region, which contained only two genes, i.e., FBXL18 and ACTB. Our finding is useful to perform a more accurate genotype-phenotype correlation and strongly strengthen the hypothesis that haploinsufficiency of ACTB is the main cause of the clinical phenotype observed in the patients with 7p22.1 microdeletions, facilitating genetic diagnosis and counseling.

Clinical and molecular characterization of a second family with the 12q14 microdeletion syndrome and review of the literature.

The 12q14 microdeletion syndrome is a rare condition characterized by low birth weight, failure to thrive, short stature, learning disabilities, and osteopoikilosis. To date, 20 cases of 12q14 deletion have been reported in the literature, displaying both phenotypic than genetic variability. We report on three familial cases, a mother and two brothers, with severe short stature. The mother and elder brother presented with osteopoikilosis while the younger brother had severe short stature and developmental delay. SNP array analysis revealed a 1.9 Mb heterozygous 12q14.2q14.3 deletion in all three patients encompassing 14 genes and 3 miRNAs. In addition, the younger brother carried a paternal 11q13.4 duplication including the SHANK2 gene. This latter patient was investigated for developmental delay and did not show osteopoikilosis, confirming the role of age in the clinical presentation of this condition. To the best of our knowledge, this is the second family described with the syndrome. Comparing the clinical and molecular data of our patients with those previously reported we performed a detailed genotype-phenotype correlation confirming the association between growth retardation and osteopoikilosis when the rearrangement includes both LEMD3 and HMGA2 genes. In addition, we suggest the XPOT, TBK1, WIF1 genes as candidates for the clinical features observed in our patients and discuss for the first time the possible involvement of some microRNAs, when deleted, in the etiology of the phenotypes in 12q14 microdeletion syndrome patients. We expect the interpretation of our findings to be useful both from a molecular point of view and for genetic counseling.

Putative TMPRSS3/GJB2 digenic inheritance of hearing loss detected by targeted resequencing.

The paper describes a putative digenic form of deafness in two siblings affected by non-syndromic hereditary hearing loss, detected by a Targeted resequencing approach. Given that a previous paper suggested TMPRSS3 and GJB2 genes as responsible for a digenic form of hearing loss, our data support and reinforce this hypothesis.

Clinical and molecular characterization of a de novo 19p13.3 microdeletion.

BACKGROUND: Structural rearrangements of chromosome 19p13.3 are a rare condition, and their phenotypic consequences remain not well defined, because of the variability of clinical manifestations. Increasing knowledge of new 19p13.3 microdeletion is useful to clarify the phenotypic variability observed in some patients. In a small number of recent papers, patients with intellectual disabilities, multiple congenital anomalies and microdeletion of the chromosome band 19p13.3 have been described. However, little is known about genes responsible for clinical features in patients carriers of 19p13.3 microdeletion; thus, increasing number of reported cases will be helpful to investigate the contribution of candidate genes, providing bases for future investigations. CASE PRESENTATION: Here, we report on a 10-years-old girl referred to our genetics clinic due to intellectual disability, attention deficit, behavioral and speech delay, hypotonia, facial dysmorphisms, eye anomalies and congenital malformations. Using an high resolution SNP array, we identified a de novo microdeletion of chromosome 19p13.3, resulting in the heterozygous loss of 27 RefSeq genes and a miRNA, partially overlapping with three others deletions already reported in literature, but extending downstream (centromeric) for additional 386 Kb. This chromosomal region includes 13 genes amongst of which we suggest for the first time the APC2, PLK5 and MBD3 genes as potential functional candidates for neurodevelopmental and behavioral phenotypes observed. CONCLUSIONS: Here we describe a patient with a 19p13.3 microdeletion that spans to the downstream chromosomal region with respect to the overlapping deletions previously reported in several other cases. The neurobehavioral features observed in our case has extended the phenotypic spectrum associated with the 19p13.3 microdeletion. New candidate genes are proposed for the neurobehavioral phenotype observed in our case.

Calcium-sensing-related gene mutations in hypercalcaemic hypocalciuric patients as differential diagnosis from primary hyperparathyroidism: detection of two novel inactivating mutations in an Italian population.

BACKGROUND: Inactivating mutations of the calcium-sensing receptor (CaSR), of the G-protein subunit α11 (GNA11) and of the adaptor-related protein complex 2, sigma 1 subunit (AP2S1) genes are responsible for familial hypocalciuric hypercalcaemia (FHH). The aim of this study was to analyse prevalence and pathogenicity of CaSR, GNA11 and AP2S1 mutations in patients with an FHH phenotype and to compare them with a sample of patients with primary hyperparathyroidism (PHPT) in order to identify the most useful laboratory parameter for a differential diagnosis. METHODS: Patients with an FHH phenotype were studied with polymerase chain reaction amplification and direct sequencing of the entire CaSR, GNA11 and AP2S1 coding sequences. Novel mutations were introduced in a Myc-tagged human wild-type (WT) CaSR cDNA-expressing vector, and functional assay was performed on human embryonic kidney cells evaluating expression and function of mutated proteins. RESULTS: Among 16 FHH patients, none had an inactivating GNA11 or AP2S1 mutation while 3 (18.8%) carried a CaSR mutation and 10 (62.5%) at least one CaSR polymorphism. Within the latter group, 7 of 10 patients had more than one polymorphism (4.1 ± 2.1 per patient). Two novel CaSR mutations [c.2120A>T (E707V) and c.2320G>A (G774S)] were identified: the E707V mutation prevented CaSR expression (western blot), whereas the G774S mutation determined a reduced receptor sensitivity to calcium (IP3 assay). PHPT patients showed significantly (P < 0.001) higher serum calcium, parathyroid hormone, urinary calcium and calcium-creatinine clearance ratio (CCCR) and significantly lower serum phosphate than FHH ones. CONCLUSIONS: FHH should be clearly differentiated by PHPT to avoid unnecessary surgery: CCCR could be a useful screening tool while genetic analysis should include the two novel CaSR mutations herein described. The role of multiple polymorphisms deserves further investigation in patients with an FHH phenotype.