Pitfalls of whole exome sequencing in undefined clinical conditions with a suspected genetic etiology.

BACKGROUND: Whole-Exome Sequencing (WES) is a valuable tool for the molecular diagnosis of patients with a suspected genetic condition. In complex and heterogeneous diseases, the interpretation of WES variants is more challenging given the absence of diagnostic handles and other reported cases with overlapping clinical presentations. OBJECTIVE: To describe candidate variants emerging from trio-WES and possibly associated with the clinical phenotype in clinically heterogeneous conditions. METHODS: We performed WES in ten patients from eight families, selected because of the lack of a clear clinical diagnosis or suspicion, the presence of multiple clinical signs, and the negative results of traditional genetic tests. RESULTS: Although we identified ten candidate variants, reaching the diagnosis of these cases is challenging, given the complexity and the rarity of these syndromes and because affected genes are already associated with known genetic diseases only partially recapitulating patients' phenotypes. However, the identification of these variants could shed light into the definition of new genotype-phenotype correlations. Here, we describe the clinical and molecular data of these cases with the aim of favoring the match with other similar cases and, hopefully, confirm our diagnostic hypotheses. CONCLUSION: This study emphasizes the major limitations associated with WES data interpretation, but also highlights its clinical utility in unraveling novel genotype-phenotype correlations in complex and heterogeneous undefined clinical conditions with a suspected genetic etiology.

From clinical to molecular diagnosis: relevance of diagnostic strategy in two cases of branchio-oto-renal syndrome - case report.

BACKGROUND: Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder characterized by deafness, branchiogenic malformations and renal abnormalities. Pathogenic variants in EYA1, SIX1 and SIX5 genes cause almost half of cases; copy number variants (CNV) and complex genomic rearrangements have been revealed in about 20% of patients, but they are not routinely and commonly included in the diagnostic work-up. CASE PRESENTATION: We report two unrelated patients with BOR syndrome clinical features, negative sequencing for BOR genes and the identification of a 2.65 Mb 8q13.2-13.3 microdeletion. CONCLUSIONS: We highlight the value of CNV analyses in high level of suspicion for BOR syndrome but negative sequencing for BOR genes and we propose an innovative diagnostic flow-chart to increase current detection rate. Our report confirms a mechanism of non-allelic homologous recombination as causing this recurrent 8q13.2-13.3 microdeletion. Moreover, considering the role of PRDM14 and NCOA2 genes, both involved in regulation of fertility and deleted in our patients, we suggest the necessity of a longer follow-up to monitor fertility issues or additional clinical findings.

Megaconial congenital muscular dystrophy due to novel CHKB variants: a case report and literature review.

BACKGROUND: Choline kinase beta (CHKB) catalyzes the first step in the de novo biosynthesis of phosphatidyl choline and phosphatidylethanolamine via the Kennedy pathway. Derangement of this pathway might also influence the homeostasis of mitochondrial membranes. Autosomal recessive CHKB mutations cause a rare form of congenital muscular dystrophy known as megaconial congenital muscular dystrophy (MCMD). CASE PRESENTATION: We describe a novel proband presenting MCMD due to unpublished CHKB mutations. The patient is a 6-year-old boy who came to our attention for cognitive impairment and slowly progressive muscular weakness. He was the first son of non-consanguineous healthy parents from Sri Lanka. Neurological examination showed proximal weakness at four limbs, weak osteotendinous reflexes, Gowers' maneuver, and waddling gate. Creatine kinase levels were mildly increased. EMG and brain MRI were normal. Left quadriceps skeletal muscle biopsy showed a myopathic pattern with nuclear centralizations and connective tissue increase. Histological and histochemical staining suggested subsarcolemmal localization and dimensional increase of mitochondria. Ultrastructural analysis confirmed the presence of enlarged ("megaconial") mitochondria. Direct sequencing of CHKB identified two novel defects: the c.1060G > C (p.Gly354Arg) substitution and the c.448-56_29del intronic deletion, segregating from father and mother, respectively. Subcloning of RT-PCR amplicons from patient's muscle RNA showed that c.448-56_29del results in the partial retention (14 nucleotides) of intron 3, altering physiological splicing and transcript stability. Biochemical studies showed reduced levels of the mitochondrial fission factor DRP1 and the severe impairment of mitochondrial respiratory chain activity in patient's muscle compared to controls. CONCLUSIONS: This report expands the molecular findings associated with MCMD and confirms the importance of considering CHKB variants in the differential diagnosis of patients presenting with muscular dystrophy and mental retardation. The clinical outcome of MCMD patients seems to be influenced by CHKB molecular defects. Histological and ultrastructural examination of muscle biopsy directed molecular studies and allowed the identification and characterization of an intronic mutation, usually escaping standard molecular testing.

Maternal Uniparental Disomy of Chromosome 20 (UPD(20)mat) as Differential Diagnosis of Silver Russell Syndrome: Identification of Three New Cases.

Silver Russell Syndrome (SRS, MIM #180860) is a rare growth retardation disorder in which clinical diagnosis is based on six features: pre- and postnatal growth failure, relative macrocephaly, prominent forehead, body asymmetry, and feeding difficulties (Netchine-Harbison clinical scoring system (NH-CSS)). The molecular mechanisms consist in (epi)genetic deregulations at multiple loci: the loss of methylation (LOM) at the paternal H19/IGF2:IG-DMR (chr11p15.5) (50%) and the maternal uniparental disomy of chromosome 7 (UPD(7)mat) (10%) are the most frequent causes. Thus far, about 40% of SRS remains undiagnosed, pointing to the need to define the rare mechanisms in such a consistent fraction of unsolved patients. Within a cohort of 176 SRS with an NH-CSS ≥ 3, a molecular diagnosis was disclosed in about 45%. Among the remaining patients, we identified in 3 probands (1.7%) with UPD(20)mat (Mulchandani-Bhoj-Conlin syndrome, OMIM #617352), a molecular mechanism deregulating the GNAS locus and described in 21 cases, characterized by severe feeding difficulties associated with failure to thrive, preterm birth, and intrauterine/postnatal growth retardation. Our patients share prominent forehead, feeding difficulties, postnatal growth delay, and advanced maternal age. Their clinical assessment and molecular diagnostic flowchart contribute to better define the characteristics of this rare imprinting disorder and to rank UPD(20)mat as the fourth most common pathogenic molecular defect causative of SRS.

Family burden of children suffering from epidermolysis bullosa.

BACKGROUND: Living with a rare disease has profound effects on the patient's life and that of their entire family, with practical and psychosocial consequences. This is particularly true when the patient is a child. The principal aim of this study was to measure the family burden in Epidermolysis Bullosa (EB). The secondary endpoint was to evaluate the possible correlation between family burden and the severity of EB. METHODS: A sample of 50 families with one or two children affected by EB were recruited between January 2016 and February 2017 to answer a 20-item questionnaire - the EB Burden of Disease (EB-BoD) - developed and validated to assess the family burden of children with EB. RESULTS: The presence of a child with EB may have profound negative implications on several different areas of daily life. In particular, the results demonstrate important differences between the different subtypes of epidermolysis bullosa regarding most of the categories considered by the questionnaire. For three categories out of four (family life, child's life, economic and social impact), a higher score is observed for children with the more debilitating forms of EB: recessive dystrophic EB (RDEB) and junctional EB (JEB). CONCLUSIONS: It is important to work with patients and their families to identify and strengthen adaptive and coping behaviors. That is possible only through the synergistic working of a multidisciplinary team made up of experienced doctors, psychologists, and social workers while in contact with patient Associations.

Olfactory Malformations in Mendelian Disorders of the Epigenetic Machinery.

Usually overlooked by physicians, olfactory abnormalities are not uncommon. Olfactory malformations have recently been reported in an emerging group of genetic disorders called Mendelian Disorders of the Epigenetic Machinery (MDEM). This study aims to determine the prevalence of olfactory malformations in a heterogeneous group of subjects with MDEM. We reviewed the clinical data of 35 patients, 20 females and 15 males, with a mean age of 9.52 years (SD 4.99). All patients had a MDEM and an already available high-resolution brain MRI scan. Two experienced neuroradiologists reviewed the MR images, noting abnormalities and classifying olfactory malformations. Main findings included Corpus Callosum, Cerebellar vermis, and olfactory defects. The latter were found in 11/35 cases (31.4%), of which 7/11 had Rubinstein-Taybi syndrome (RSTS), 2/11 had CHARGE syndrome, 1/11 had Kleefstra syndrome (KLFS), and 1/11 had Weaver syndrome (WVS). The irregularities mainly concerned the olfactory bulbs and were bilateral in 9/11 patients. With over 30% of our sample having an olfactory malformation, this study reveals a possible new diagnostic marker for MDEM and links the epigenetic machinery to the development of the olfactory bulbs.

Simultaneous Detection of NF1, SPRED1, LZTR1, and NF2 Gene Mutations by Targeted NGS in an Italian Cohort of Suspected NF1 Patients.

Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene; we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients; the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed; in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.

Unexpected phenotype in a frameshift mutation of PTCH1.

BACKGROUND: Gorlin syndrome, also known as basal cell nevus syndrome (BCNS), is a rare autosomal dominant genetic condition, characterized by the presence of multiple basal cell carcinomas at a young age, odontogenic keratocysts, skeletal anomalies, macrocephaly, and dysmorphisms. BCNS is mainly caused by mutations in PTCH1, an onco-suppressor gene that maps at 9q22.3 region. A disease related to BCNS is the 9q22.3 microdeletion syndrome. This condition has an overlapping clinical phenotype with the BCNS, but it can present in addition: metopic craniosynostosis, overgrowth, obstructive hydrocephalus, developmental delay, intellectual disability, and seizures. This syndrome is caused by the deletion of a genomic region containing the PTCH1 and the FANCC. METHODS AND RESULTS: We report the case of an 11-year-old girl that came to our attention for overgrowth, dysmorphic features of the face, and craniosynostosis, but with a normal intellectual and motor development. At first we performed an array-comparative genomic hybridization (aCGH) analysis. The analysis showed no copy number changes. Then, we performed the analysis of the PTCH1 by next-generation sequencing. This analysis showed a heterozygous frameshift mutation. CONCLUSION: This is the first case with a PTCH1 point mutation with a 9q22.3 microdeletion syndrome phenotype. This finding may strengthen the importance of the role of the PTCH1, especially regarding the metopic craniosynostosis.

Risk of Optic Pathway Glioma in Neurofibromatosis Type 1: No Evidence of Genotype-Phenotype Correlations in A Large Independent Cohort.

The occurrence of optic pathway gliomas (OPGs) in children with neurofibromatosis type 1 (NF1) still raises many questions regarding screening and surveillance because of the lack of robust prognostic factors. Recent studies of an overall cohort of 381 patients have suggested that the genotype may be the main determinant of the development of OPG, with the risk being higher in patients harbouring NF1 mutations in the 5' tertile and the cysteine/serine-rich domain. In an attempt to confirm this hypothesis, we used strict criteria to select a large independent cohort of 309 NF1 patients with defined constitutional NF1 mutations and appropriate brain images (255 directly enrolled and 54 as a result of a literature search). One hundred and thirty-two patients had OPG and 177 did not. The association of the position (tertiles and functional domains) and type of NF1 mutation with the development of OPG was analysed using the χ2 test and Fisher's exact probability test; odds ratios (ORs) with 95% confidence intervals were calculated, and Bonferroni's correction for multiple comparisons was applied; multiple logistic regression was also used to study genotype-phenotype associations further. Our findings show no significant correlation between the site/type of NF1 mutation and the risk of OPG, and thus do not support the hypothesis that certain constitutional mutations provide prognostic information in this regard. In addition, we combined our cohort with a previously described cohort of 381 patients for a total of 690 patients and statistically re-analysed the results. The re-analysis confirmed that there were no correlations between the site (tertile and domain) and the risk of OPG, thus further strengthening our conclusions.

New Insights into Kleefstra Syndrome: Report of Two Novel Cases with Previously Unreported Features and Literature Review.

Kleefstra syndrome (KS) is a rare genetic condition resulting from either 9q34.3 microdeletions or mutations in the EHMT1 gene located in the same genomic region. To date, approximately 100 patients have been reported, thereby allowing the core phenotype of KS to be defined as developmental delay/intellectual disability, generalized hypotonia, neuropsychiatric anomalies, and a distinctive facial appearance. Here, to further expand the knowledge on genotype and phenotype of this condition, we report 2 novel cases: one patient carrying a 46-kb 9q34.3 deletion and showing macrocephaly never described in KS, and a second patient carrying a classic 9q34.3 deletion, presenting with a previously unreported skeletal feature (postaxial polydactyly of the right foot) and an unusual brain anomaly (olfactory bulb hypoplasia) observed via magnetic resonance imaging. Further, we provide a review of the current literature regarding KS and compare these 2 patients with those previously described, thereby confirming that the genotype-phenotype correlation in KS remains difficult to determine.

A novel splice site variant in ITPR1 gene underlying recessive Gillespie syndrome.

Gillespie syndrome (GLSP) is a rare congenital disorder characterized by partial aniridia, hypotonia, progressive cerebellar hypoplasia, nonprogressive ataxia, and intellectual disability. All causative variants to date affect the central or the 3'-terminal domains of ITPR1 gene and exhibit autosomal recessive or dominant inheritance pattern. We investigated by exome sequencing the molecular cause of GLSP in a family composed by consanguineous healthy parents, two affected siblings and one healthy son. We found the novel splice site variant c.278_279 + 2delACGT located at the 5'-end of ITPR1. The affected siblings were homozygotes, their parents heterozygous carriers and the variant was absent in the healthy son, indicating a recessive inheritance pattern. The deletion abolished the splice-donor site at exon 5/intron 5 junction, causing the skipping of exon 5 and the generation of a premature STOP codon. The mutation is predicted to result in the synthesis of a 64-amino acids nonfunctional protein. The mutant transcript comprised >96% of ITPR1 mRNA in the affected siblings, indicating that a small amount of wild-type transcript was still present. The novel autosomal recessive mutation here reported is the first variant affecting the ITPR1 N-terminal suppressor domain, thus extending the spectrum of the pathogenetic variants in GLSP and the range of the associated clinical manifestations.

Perthes disease: A new finding in Floating-Harbor syndrome.

Floating-Harbor Syndrome (FHS; OMIM #136140) is an ultra-rare autosomal dominant genetic condition characterized by expressive language delay, short stature with delayed bone mineralization, a triangular face with a prominent nose, and deep-set eyes, and hand anomalies. First reported in 1973, FHS is associated with mutations in the SRCAP gene, which encodes SNF2-related CREBBP activator protein. Mutations in the CREBBP gene cause Rubinstein-Taybi Syndrome (RSTS; OMIM #180849, #613684), another rare disease characterized by broad thumbs and halluces, facial dysmorphisms, short stature, and intellectual disability, which has a phenotypic overlap with FHS. We describe a case of FHS associated with a novel SRCAP mutation and characterized by Perthes disease, a skeletal anomaly described in approximately 3% of patients with RSTS. Thus Perthes disease can be added to the list of clinical features that overlap between FHS and RSTS.

Correction: The absence that makes the difference: choroidal abnormalities in Legius syndrome.

Correction to: Journal of Human Genetics advance online publication 27 July 2017; https://doi.org/10.1038/jhg.2017.78.

Identification of an atypical microdeletion generating the RNF135-SUZ12 chimeric gene and causing a position effect in an NF1 patient with overgrowth.

Neurofibromatosis type I (NF1) microdeletion syndrome, which is present in 4-11% of NF1 patients, is associated with a severe phenotype as it is caused by the deletion of NF1 and other genes in the 17q11.2 region. The variable expressivity of the disease makes it challenging to establish genotype-phenotype correlations, which also affects prognosis and counselling. We here describe a 3-year-old NF1 patient with an atypical deletion and a complex phenotype. The patient showed overgrowth, café au lait spots, inguinal freckling, and neurological abnormalities. The extent of the deletion was determined by means of array comparative genomic hybridisation, and its breakpoints were isolated by means of long-range polymerase chain reaction. Sequence analysis of the deletion junction fragment revealed the occurrence of an Alu-mediated recombination that led to the generation of a chimeric gene consisting of three exons of RNF135 and eleven exons of SUZ12. Interestingly, the deletion shares a common RNF135-centred region with another deletion described in a non-NF1 patient with overgrowth. In comparison with the normal RNF135 allele, the chimeric transcript was 350-fold over-expressed in peripheral blood, and the ADAP2 gene located upstream of RNF135 was also up-regulated. In line with this, the deletion causes the loss of a chromatin TD boundary, which entails the aberrant adoption of distal cis-acting regulatory elements. These findings suggest that RNF135 haploinsufficiency is related to overgrowth in patients with NF1 microdeletion syndrome and, for the first time, strongly indicate a position effect that warrants further genotype-phenotype correlation studies to investigate the possible existence of previously unknown pathogenic mechanisms.

The absence that makes the difference: choroidal abnormalities in Legius syndrome.

Neurofibromatosis type 1 (NF1) is an hereditary disorder characterized by abnormal proliferation of multiple tissues of neural crest origin, and presents mainly with multiple café-au-lait macules, axillary freckling and neurofibromas. Choroidal involvement in NF1 patients has been studied, thanks to the development of non-invasive tools such as infrared monochromatic light during fundus examination, which showed bright patchy lesions consistent with choroidal nodules. Choroidal abnormalities identified with near-infrared reflectance have reported with a frequency of up to 100% in NF1, and have been recently been proposed as a novel diagnostic criterion for NF1. Legius syndrome can be clinically indistinguishable from NF1 and results in a small percentage of individuals being misdiagnosed. We investigated the presence of choroidal abnormalities in Legius syndrome to determine their specificity to NF1 and their potential usefulness as a novel diagnostic criterion for NF1. We examined the fundus of 16 eyes by confocal scanning laser ophthalmoscopy with infrared monochromatic light in eight patients with molecularly confirmed Legius syndrome. No abnormalities were observed, confirming the diagnostic value of choroidal abnormalities for the diagnosis of NF1.

16p13 microduplication without CREBBP involvement: Moving toward a phenotype delineation.

The short arm of chromosome 16 is one of the less stable regions of our genome, as over 10% of the euchromatic region of 16p is composed of highly complex low copy repeats that are known to be predisposed to rearrangements mediated by non-allelic homologous recombination. The 16p13.3p13.13 molecular region has been defined as the 16p duplication hotspot, and duplications of chromosome 16p13 have recently been confirmed to cause a recognizable syndrome, with CREBBP being the main phenotype-causing gene. To date, only one case report is present in the literature with a 16p13 duplication without CREBBP involvement; we describe here a second analogous case with a not previously reported 16p13.2p13.13 microduplication. This paper allows us to better delineate the clinical features of 16p13 microduplications that do not encompass CREBBP and, concurrently, to narrow the molecular region responsible for congenital heart defects in 16p duplications as well as to propose GRIN2A as a candidate gene for epilepsy.

Is cutis verticis Gyrata-Intellectual Disability syndrome an underdiagnosed condition? A case report and review of 62 cases.

Cutis Verticis Gyrata-Intellectual Disability (CVG-ID) syndrome is a rare neurocutaneous syndrome characterized by intellectual disability and scalp folds and furrows that are typically absent at birth and are first noticed after puberty. First reported in 1893, the syndrome was mainly identified in subjects living in psychiatric institutions, where it was found to have a prevalence of up to 11.4%. Most patients were reported in the literature during the first half of the 20th century. CVG-ID is now a less reported and possibly under-recognized syndrome. Here, we report a patient with CVG-ID that was diagnosed using the novel approach of magnetic resonance imaging and we conduct a systematic review of all patients reported in the last 60 years, discussing the core clinical features of this syndrome. © 2016 Wiley Periodicals, Inc.

MIR137 is the key gene mediator of the syndromic obesity phenotype of patients with 1p21.3 microdeletions.

BACKGROUND: Deletions in the long arm of chromosome 1 have been described in patients with a phenotype consisting primarily of obesity, intellectual disability and autism-spectrum disorder. The minimal region of overlap comprises two genes: DPYD and MIR137. CASE PRESENTATION: We describe a 10-year-old boy with syndromic obesity who carries a novel 1p21.3 deletion overlapping the critical region with the MIR137 gene only. CONCLUSIONS: This study suggests that MIR137 is the mediator of the obesity phenotype of patients carrying 1p21.3 microdeletions.

Autoimmunity and Cytokine Imbalance in Inherited Epidermolysis Bullosa.

In order to evaluate the serum anti-skin autoantibodies and cytokine concentrations in patients with different epidermolysis bullosa (EB) types and severity, 42 EB patients and 38 controls were enrolled. Serum anti-skin antibodies were significantly higher in the patients than in the controls (p = 0.008, p < 0.001, p < 0.001, p < 0.001 and p < 0.001 for desmoglein 1 (DSG1) desmoglein 3 (DSG3), bullous pemphigoid 180 (BP180), BP230 and type VII collagen (COL7), respectively). The same trend was observed for interleukin (IL)-1ß, IL-2, IL-6, IL-10, tumor necrosis factor-ß, and interferon-γ (p < 0.001, p < 0.001, p < 0.001, p = 0.008, p < 0.001 and p = 0.002, respectively). Increases in anti-skin antibodies and cytokine concentrations were higher in patients with recessive dystrophic EB than in those with different types of EB, in generalized cases than in localized ones, and in patients with higher Birmingham Epidermolysis Bullosa Severity (BEBS) scores than in those with a lower score. The BEBS score was directly correlated with BP180, BP230, COL7 (p = 0.015, p = 0.008 and p < 0.001, respectively) and IL-6 (p = 0.03), whereas IL-6 appeared significantly associated with DSG1, DSG3, BP180, BP230 and COL7 (p = 0.015, p = 0.023, p = 0.023, p = 0.015 and p = 0.005, respectively). This study showed that autoimmunity and inflammatory responses are frequently activated in EB, mainly in severe forms, suggesting the use of immunosuppressive drugs or biologicals that are active against pro-inflammatory cytokines to reduce clinical signs and symptoms of disease.