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.

Opitz syndrome: improving clinical interpretation of intronic variants in MID1 gene.

BACKGROUND: Loss-of-function variants in MID1 are the most common cause of Opitz G/BBB syndrome (OS). The interpretation of intronic variants affecting the splicing is a rising issue in OS. METHODS: Exon sequencing of a 2-year-old boy with OS showed that he was a carrier of the de novo c.1286-10G>T variant in MID1. In silico predictions and minigene assays explored the effect of the variant on splicing. The minigene approach was also applied to two previously identified MID1 c.864+1G>T and c.1285+1G>T variants. RESULTS: Minigene assay demonstrated that the c.1286-10G>T variant generated the inclusion of eight nucleotides that predicted generation of a frameshift. The c.864+1G>T and c.1285+1G>T variants resulted in an in-frame deletion predicted to generate a shorter MID1 protein. In hemizygous males, this allowed reclassification of all the identified variants from "of unknown significance" to "likely pathogenic." CONCLUSIONS: Minigene assay supports functional effects from MID1 intronic variants. This paves the way to the introduction of similar second-tier investigations in the molecular diagnostics workflow of OS. IMPACT: Causative intronic variants in MID1 are rarely investigated in Opitz syndrome. MID1 is not expressed in blood and mRNA studies are hardly accessible in routine diagnostics. Minigene assay is an alternative for assessing the effect of intronic variants on splicing. This is the first study characterizing the molecular consequences of three MID1 variants for diagnostic purposes and demonstrating the efficacy of minigene assays in supporting their clinical interpretation. Review of the criteria according to the American College of Medical Genetics reassessed all variants as likely pathogenic.

A novel homozygous variant in COX5A causes an attenuated phenotype with failure to thrive, lactic acidosis, hypoglycemia, and short stature.

Genetic defect in the nuclear encoded subunits of cytochrome c oxidase are very rare. To date, most deleterious variants affect the mitochondrially encoded subunits of complex IV and the nuclear genes encoded for assembly factors. A biallelic pathogenic variant in the mitochondrial complex IV subunit COX5A was previously reported in a couple of sibs with failure to thrive, lactic acidosis and pulmonary hypertension and a lethal phenotype. Here, we describe a second family with a 11-year-old girl presenting with failure to thrive, lactic acidosis, hypoglycemia and short stature. Clinical exome revealed the homozygous missense variant c.266 T > G in COX5A, which produces a drop of the corresponding protein and a reduction of the COX activity. Compared to the previous observation, this girl showed an attenuated metabolic derangement without involvement of the cardiovascular system and neurodevelopment. Our observation confirms that COX5A recessive variants may cause mitochondrial disease and expands the associated phenotype to less severe presentations.

Resequencing of candidate genes for Keratoconus reveals a role for Ehlers-Danlos Syndrome genes.

The involvement of genetic factors in the pathogenesis of KC has long been recognized but the identification of variants affecting the underlying protein functions has been challenging. In this study, we selected 34 candidate genes for KC based on previous whole-exome sequencing (WES) and the literature, and resequenced them in 745 KC patients and 810 ethnically matched controls from Belgium, France and Italy. Data analysis was performed using the single variant association test as well as gene-based mutation burden and variance components tests. In our study, we detected enrichment of genetic variation across multiple gene-based tests for the genes COL2A1, COL5A1, TNXB, and ZNF469. The top hit in the single variant association test was obtained for a common variant in the COL12A1 gene. These associations were consistently found across independent subpopulations. Interestingly, COL5A1, TNXB, ZNF469 and COL12A1 are all known Ehlers-Danlos Syndrome (EDS) genes. Though the co-occurrence of KC and EDS has been reported previously, this study is the first to demonstrate a consistent role of genetic variants in EDS genes in the etiology of KC. In conclusion, our data show a shared genetic etiology between KC and EDS, and clearly confirm the currently disputed role of ZNF469 in disease susceptibility for KC.

Novel TONSL variants cause SPONASTRIME dysplasia and associate with spontaneous chromosome breaks, defective cell proliferation and apoptosis.

SPONASTRIME dysplasia is an ultrarare spondyloepimetaphyseal dysplasia featuring short stature and short limbs, platyspondyly, depressed nasal bridge with midface hypoplasia and striated metaphyses. In 2019, an autosomal recessive inheritance was demonstrated by the identification of bi-allelic hypomorphic alleles in TONSL. The encoded protein has a critical role in maintaining genome integrity by promoting the homologous recombination required for repairing spontaneous replication-associated DNA lesions at collapsed replication forks. We report a 9-year-old girl with typical SPONASTRIME dysplasia and resulted in carrier of the novel missense p.(Gln430Arg) and p.(Leu1090Arg) variants in TONSL at whole-exome sequencing. In silico analysis predicted that these variants induced thermodynamic changes with a pathogenic impact on protein function. To support the pathogenicity of the identified variants, cytogenetic analysis and microscopy assays showed that patient-derived fibroblasts exhibited spontaneous chromosomal breaks and flow cytometry demonstrated defects in cell proliferation and enhanced apoptosis. These findings contribute to our understanding of the molecular pathogenesis of SPONASTRIME dysplasia and might open the way to novel therapeutic approaches.

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.

Mitochondrial DNA copy number in affected and unaffected LHON mutation carriers.

OBJECTIVES: Leber's hereditary optic neuropathy (LHON) is a mitochondrial genetic disease characterized by a variable and reduced penetrance. Individuals carrying a primary LHON-causing mitochondrial DNA (mtDNA) mutation may either remain asymptomatic lifelong, as unaffected carriers, or develop sudden central visual loss that rapidly aggravates over some weeks. Over the years several genetic/environmental triggers able to modulate the risk of developing LHON have been proposed. We provided data supporting a possible correlation between LHON penetrance and the mtDNA copy number, a raw index of mitochondrial mass, whose increase could represent a compensatory response that cells implement to alleviate the pathogenic effect of the primary LHON-causing mtDNA mutations. DATA DESCRIPTION: We collected Italian and Spanish subjects harboring one of the three common LHON primary mutations, either in heteroplasmic or homoplasmic status. For each population we were able to discriminate between affected subjects presenting typical clinical tracts of LHON and LHON-causing mutation carriers showing no symptoms correlated with vision loss. Each subject has been characterized for the presence of a LHON primary mutation, for its status of homoplasmy or heteroplasmy, and for the mtDNA content per cell, expressed as relative mtDNA/nDNA ratio respect to controls. Additional clinical information is present for all the Italian subjects.

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.

A single-center study on 140 patients with cerebral cavernous malformations: 28 new pathogenic variants and functional characterization of a PDCD10 large deletion.

Cerebral cavernous malformation (CCM) is a capillary malformation arising in the central nervous system. CCM may occur sporadically or cluster in families with autosomal dominant transmission, incomplete penetrance, and variable expressivity. Three genes are associated with CCM KRIT1, CCM2, and PDCD10. This work is a retrospective single-center molecular study on samples from multiple Italian clinical providers. From a pool of 317 CCM index patients, we found germline variants in either of the three genes in 80 (25.2%) probands, for a total of 55 different variants. In available families, extended molecular analysis found segregation in 60 additional subjects, for a total of 140 mutated individuals. From the 55 variants, 39 occurred in KRIT1 (20 novel), 8 in CCM2 (4 novel), and 8 in PDCD10 (4 novel). Effects of the three novel KRIT1 missense variants were characterized in silico. We also investigated a novel PDCD10 deletion spanning exon 4-10, on patient's fibroblasts, which showed significant reduction of interactions between KRIT1 and CCM2 encoded proteins and impaired autophagy process. This is the largest study in Italian CCM patients and expands the known mutational spectrum of KRIT1, CCM2, and PDCD10. Our approach highlights the relevance of seeking supporting information to pathogenicity of new variants for the improvement of management of CCM.

Leber's hereditary optic neuropathy, intellectual disability and epilepsy presenting with variable penetrance associated to the m.3460G >A mutation and a heteroplasmic expansion of the microsatellite in MTRNR1 gene - case report.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) associated with mutations in mitochondrial DNA (mtDNA) typically manifests only optic nerve involvement but in some patients may develop additional neurological complications. The cause of this association is not clear. CASE PRESENTATION: We present a case of a 24-year-old male with a history of subacute, painless, and rapidly progressive bilateral vision loss. We performed ophthalmological, neurological and neuropsychological investigations in the proband and his LHON family. The proband showed optic neuropathy, epilepsy, migraine, and intellectual disability; all the maternal relatives did not manifest optic neuropathy but a moderate to severe intellectual disability. Genetic screening revealed a novel association of the LHON m.3460G > A primary mutation with the m.T961delT + C(n)ins within the mitochondrial encoded 12S RNA (MTRNR1) gene which segregates with the intellectual disability through the maternal branch of the family. We also found a significant increase of mtDNA content in all the unaffected homo/heteroplasmic mutation carriers with respect to either affected or control subjects. CONCLUSION: This is the first case reporting the co-segregation of a mutation in MTRNR1 gene with a LHON primary mutation, which may be a risk factor of the extraocular signs complicating LHON phenotype. In addition, the data herein reported, confirmed that the key factor modulating the penetrance of optic atrophy in the family is the amount of mtDNA.

High Mitochondrial DNA Copy Number Is a Protective Factor From Vision Loss in Heteroplasmic Leber's Hereditary Optic Neuropathy (LHON).

PURPOSE: Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease that typically causes bilateral blindness in young men. It is characterized by as yet undisclosed genetic and environmental factors affecting the incomplete penetrance. METHODS: We identified 27 LHON subjects who possess heteroplasmic primary LHON mutations. Mitochondrial DNA (mtDNA) copy number was evaluated. RESULTS: The presence of centrocecal scotoma, an edematous, hyperemic optic nerve head, and vascular tortuosity, as well as telangiectasia was recognized in affected subjects. We found higher cellular mtDNA content in peripheral blood cells of unaffected heteroplasmic mutation carriers with respect to the affected. CONCLUSIONS: The increase of cellular mtDNA content prevents complete loss of vision despite the presence of a heteroplasmic state of LHON primary mutation, suggesting that it is a key factor responsible for penetrance of LHON.

Leber's hereditary optic neuropathy (LHON) in an Apulian cohort of subjects.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder that causes severe loss of sight in young adults, and is typically associated to mitochondrial DNA (mtDNA) mutations. Heteroplasmy of primary LHON mutations, presence of 'ancillary' mtDNA mutations, and mtDNA copy number are probably correlated with the penetrance and the severity of the disease. In this study, we performed a mutational screening in an Apulian cohort of LHON patients and we found that 41 out of 54 subjects harbored the m.11778G>A mutation, and 13 harbored the m.3460G>A mutation. Whole mtDNA sequencing was performed in three affected subjects belonging to three unrelated m.11778G>A pedigrees to evaluate the putative synergistic role of additional mtDNA mutations in determining the phenotype. Our study suggests to include haplogroup T as a possible genetic background influencing LHON penetrance and to consider the increase of mtDNA copy number as a protective factor from vision loss regardless the hetero/homoplasmic status of LHON primary mutations.

Expanding the mutation spectrum in 130 probands with ARPKD: identification of 62 novel PKHD1 mutations by sanger sequencing and MLPA analysis.

Autosomal recessive polycystic kidney disease (ARPKD) is a rare severe genetic disorder arising in the perinatal period, although a late-onset presentation of the disease has been described. Pulmonary hypoplasia is the major cause of morbidity and mortality in the newborn period. ARPKD is caused by mutations in the PKHD1 (polycystic kidney and hepatic disease 1) gene that is among the largest human genes. To achieve a molecular diagnosis of the disease, a large series of Italian affected subjects were recruited. Exhaustive mutation analysis of PKHD1 gene was carried out by Sanger sequencing and multiple ligation probe amplification (MLPA) technique in 110 individuals. A total of 173 mutations resulting in a detection rate of 78.6% were identified. Additional 20 unrelated patients, in whom it was not possible to analyze the whole coding sequence, have been included in this study. Taking into account the total number (n=130) of this cohort of patients, 107 different types of mutations have been detected in 193 mutated alleles. Out of 107 mutations, 62 were novel: 11 nonsense, 6 frameshift, 7 splice site mutations, 2 in-frame deletions and 2 multiexon deletion detected by MLPA. Thirty-four were missense variants. In conclusion, our report expands the spectrum of PKHD1 mutations and confirms the heterogeneity of this disorder. The population under study represents the largest Italian ARPKD cohort reported to date. The estimated costs and the time invested for molecular screening of genes with large size and allelic heterogeneity such as PKHD1 demand the use of next-generation sequencing (NGS) technologies for a faster and cheaper screening of the affected subjects.

Identification and Clinical Characterization of Adult Patients with Multigenerational Diabetes Mellitus.

BACKGROUND: Some patients diagnosed as having type 2 diabetes mellitus (T2DM) are, instead, affected by multigenerational diabetes whose clinical characteristics are mostly undefined. OBJECTIVE: 1. To identify among patients who had been previously defined as affected by T2DM those, in fact, affected by multigenerational diabetes; 2. After excluding patients carrying the most common MODY genes and mitochondrial mutations, we compared clinical features of remaining patients with those of patients with T2DM. METHODS: Among 2,583 consecutive adult patients who had been defined as affected by T2DM, we looked for those with diabetes in ≥3 consecutive generations. All probands were screened for mutations in six MODY genes (HNF4A, GCK, HNF1A, PDX1, HNF1B and NeuroD1) and for the A3243G mitochondrial mutation. After excluding patients with mutations in one of such genes, we compared clinical features of the remaining 67 patients (2.6% of the whole initial sample) affected by multigenerational "familial diabetes of the adulthood" (FDA) and of their diabetic relatives (n = 63) to those with T2DM (n = 1,028) by generalized hierarchical linear models followed by pairwise comparisons. RESULTS: Age, age at diagnosis, proportion of hypertension (all p<0.001), and waist circumference (p<0.05) were lower in FDA than T2DM. Nonetheless, the two groups had similar age-adjusted incidence rate of all-cause mortality. CONCLUSIONS: Beside younger age at diagnosis, FDA patients show lower waist circumference and reduced proportion of hypertension as compared to those with T2DM; despite such reduced potential cardiovascular risk factors, FDA patients did not show a reduced mortality risk than patients with T2DM.

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients.

Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been associated with early onset encephalopathy with signs of oxidative phosphorylation defects classified as pontocerebellar hypoplasia 6. We describe clinical, neuroimaging and molecular features on five patients from three unrelated families who displayed mutations in RARS2. All patients rapidly developed a neonatal or early-infantile epileptic encephalopathy with intractable seizures. The long-term follow-up revealed a virtual absence of psychomotor development, progressive microcephaly, and feeding difficulties. Mitochondrial respiratory chain enzymes in muscle and fibroblasts were normal in two. Blood and CSF lactate was abnormally elevated in all five patients at early stages while appearing only occasionally abnormal with the progression of the disease. Cerebellar vermis hypoplasia with normal aspect of the cerebral and cerebellar hemispheres appeared within the first months of life at brain MRI. In three patients follow-up neuroimaging revealed a progressive pontocerebellar and cerebral cortical atrophy. Molecular investigations of RARS2 disclosed the c.25A>G/p.I9V and the c.1586+3A>T in family A, the c.734G>A/p.R245Q and the c.1406G>A/p.R469H in family B, and the c.721T>A/p.W241R and c.35A>G/p.Q12R in family C. Functional complementation studies in Saccharomyces cerevisiae showed that mutation MSR1-R531H (equivalent to human p.R469H) abolished respiration whereas the MSR1-R306Q strain (corresponding to p.R245Q) displayed a reduced growth on non-fermentable YPG medium. Although mutations functionally disrupted yeast we found a relatively well preserved arginine aminoacylation of mitochondrial tRNA. Clinical and neuroimaging findings are important clues to raise suspicion and to reach diagnostic accuracy for RARS2 mutations considering that biochemical abnormalities may be absent in muscle biopsy.

20 novel point mutations and one large deletion in EXT1 and EXT2 genes: report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple exostosis.

BACKGROUND: Hereditary multiple exostosis represents the most frequent bone tumor disease in humans. It consists of cartilage deformities affecting the juxta-ephyseal region of long bones. Usually benign, exostosis could degenerate in malignant chondrosarcoma form in less than 5% of the cases. Being caused by mutations in the predicted tumor suppressor genes, EXT1 (chr 8q23-q24) and EXT2 (chr 11p11-p12) genes, HMEs are usually inherited with an autosomal dominant pattern, although "de novo" cases are not infrequent. AIM: Here we present our genetic diagnostic report on the largest Southern Italy cohort of HME patients consisting of 90 subjects recruited over the last 5years. RESULTS: Molecular screening performed by direct sequencing of both EXT1 and EXT2 genes, by MLPA and Array CGH analyses led to the identification of 66 mutations (56 different occurrences) and one large EXT2 deletion out of 90 patients (74.4%). The total of 21 mutations (20 different occurrences, 33.3%) and the EXT2 gene deletion were novel. In agreement with literature data, EXT1 gene mutations were scattered along all the protein sequence, while EXT2 lesions fell in the first part of the protein. Conservation, damaging prediction and 3-D modeling, in-silico, analyses, performed on three novel missense variants, confirmed that at least in two cases the novel aminoacidic changes could alter the structure stability causing a strong protein misfolding. CONCLUSIONS: Here we present 20 novel EXT1/EXT2 mutations and one large EXT2 deletion identified in the largest Southern Italy cohort of patients affected by hereditary multiple exostosis.

Mutational screening of VSX1, SPARC, SOD1, LOX, and TIMP3 in keratoconus.

PURPOSE: To evaluate the involvement of Visual System Homeobox 1 (VSX1), Secreted Protein Acidic and Rich in Cysteine (SPARC), Superoxide Dismutase 1 (SOD1), Lysyl Oxidase (LOX), and Tissue Inhibitor of Metalloproteinase 3 (TIMP3) in sporadic and familial keratoconus. METHODS: Mutational analysis of the five genes was performed by sequencing and fragment analysis in a large cohort of 302 Italian patients, with a diagnosis of keratoconus based on clinical examination and corneal topography. The variants identified in VSX1 and SPARC were also assessed in the available relatives of the probands. RESULTS: A novel mutation p.G239R and previously reported mutations were found in VSX1. Novel and already reported variants were identified in SPARC and SOD1, whose pathogenic significance has not been established. No pathogenic variants have been identified in LOX and TIMP3. CONCLUSIONS: Molecular analysis of the five genes in a cohort of 225 sporadic and 77 familial keratoconus cases confirms the possible pathogenic role of VSX1 though in a small number of patients; a possible involvement of LOX and TIMP3 could be excluded; and the role played by SOD1 and SPARC in determining the disease as not been definitively clarified. Further studies are required to identify other important genetic factors involved in the pathogenesis and progression of the disease that in the authors' opinion, and according with several authors, should be considered as a complex disease.