FOXI3 pathogenic variants cause one form of craniofacial microsomia.

Craniofacial microsomia (CFM; also known as Goldenhar syndrome), is a craniofacial developmental disorder of variable expressivity and severity with a recognizable set of abnormalities. These birth defects are associated with structures derived from the first and second pharyngeal arches, can occur unilaterally and include ear dysplasia, microtia, preauricular tags and pits, facial asymmetry and other malformations. The inheritance pattern is controversial, and the molecular etiology of this syndrome is largely unknown. A total of 670 patients belonging to unrelated pedigrees with European and Chinese ancestry with CFM, are investigated. We identify 18 likely pathogenic variants in 21 probands (3.1%) in FOXI3. Biochemical experiments on transcriptional activity and subcellular localization of the likely pathogenic FOXI3 variants, and knock-in mouse studies strongly support the involvement of FOXI3 in CFM. Our findings indicate autosomal dominant inheritance with reduced penetrance, and/or autosomal recessive inheritance. The phenotypic expression of the FOXI3 variants is variable. The penetrance of the likely pathogenic variants in the seemingly dominant form is reduced, since a considerable number of such variants in affected individuals were inherited from non-affected parents. Here we provide suggestive evidence that common variation in the FOXI3 allele in trans with the pathogenic variant could modify the phenotypic severity and accounts for the incomplete penetrance.

Phenotype-driven variant filtration strategy in exome sequencing toward a high diagnostic yield and identification of 85 novel variants in 400 patients with rare Mendelian disorders.

About 6000 to 7000 different rare disorders with suspected genetic etiologies have been described and almost 4500 causative gene(s) have been identified. The advent of next-generation sequencing (NGS) technologies has revolutionized genomic research and diagnostics, representing a major advance in the identification of pathogenic genetic variations. This study presents a 3-year experience from an academic genetics center, where 400 patients were referred for genetic analysis of disorders with unknown etiology. A phenotype-driven proband-only exome sequencing (ES) strategy was applied for the investigation of rare disorders, in the context of optimizing ES diagnostic yield and minimizing costs and time to definitive diagnosis. Overall molecular diagnostic yield reached 53% and characterized 243 pathogenic variants in 210 cases, 85 of which were novel and 148 known, contributing information to the community of disease and variant databases. ES provides an opportunity to resolve the genetic etiology of disorders and support appropriate medical management and genetic counseling. In cases with complex phenotypes, the identification of complex genotypes may contribute to more comprehensive clinical management. In the context of effective multidisciplinary collaboration between clinicians and laboratories, ES provides an efficient and appropriate tool for first-tier genomic analysis.

Bone Marrow Failure in Fanconi Anemia: Clinical and Genetic Spectrum in a Cohort of 20 Pediatric Patients.

Prognostic refinement in Fanconi anemia (FA) is needed, especially when considering allogeneic hematopoietic stem cell transplantation (HCT). We studied 20 children with FA and bone marrow failure from a single center. According to Hôpital Saint-Louis risk classification for FA, patients were classified in stage A (no or mild cytopenia/dysplasia), B (single non-high-risk cytogenetic abnormality), C (severe cytopenia and/or significant dysplasia and/or high-risk cytogenetic abnormality), and D (myelodysplastic syndrome with excess of blasts/acute myeloid leukemia) in 4, 2, 13, and 0 cases, respectively. Nine patients received androgens +/- steroids, with a response rate of 30%, and 11 patients underwent HCT. Ten-year cumulative incidence (CI) of myelodysplastic syndrome/acute myeloid leukemia and overall survival (OS) were 21.9% and 45.3%, respectively, in the entire cohort, whereas cumulative incidence of transplantation-related mortality and OS were 27% and 63%, respectively, in patients who underwent HCT. Patients with significant dysplasia at diagnosis (stages C and D) had significantly shorter OS post-HCT as compared with patients without dysplasia. All patients in stages C and D at diagnosis or during evolution died from their disease. HCT in recent years was associated with more favorable outcomes. Larger cohorts could validate homogenous reporting of risk and help decision-making, particularly for HCT.

The genetic etiology in cerebral palsy mimics: The results from a Greek tertiary care center.

OBJECTIVE: Non-progressive genetic disorders may present with motor dysfunction resembling cerebral palsy (CP). Such patients are often characterized as CP mimics. The purpose of this work was to delineate the clinical manifestations and molecular findings of CP mimic patients, with the ultimate goal to offer specific disease-modifying therapy and genetic counseling. METHODS: Retrospective study of 47 patients diagnosed with CP and no acquired etiology. Chart review of clinical, neuroradiological, biochemical and molecular data was performed. RESULTS: 31,91% of patients manifested with features resembling dyskinetic CP, 19,14% spastic CP, 10,63% ataxic CP and 38,30% mixed CP. In 23 patients molecular diagnosis was reached and included 5 hereditary spastic paraplegia genes (SPG) in spastic CP mimics; HPRT1, TH, QDPR, DDC in dystonic CP mimics; ADCY5 and NIKX2-1 in choreic CP mimics; CANA1A in ataxic CP mimics; and SPG, PDHA1, NIKX2-1, AT, SLC2A1 and SPR in mixed CP mimics. In 14 patients, the etiological diagnosis led to specific treatment. CONCLUSIONS: CP mimics show a number of features that differ from classic CP and can be used as diagnostic clues, including presence of mixed motor features, minor dysmorphic features, oculogyric movements, multiple features of autonomic dysfunction, and acquired microcephaly. A more stringent use of the concept of CP focused on acquired lesions during the perinatal and infancy periods, and excluding disorders that could be of genetic origin, could contribute to a purer use of the term. Identification of a specific genetic cause for CP mimics may in certain cases lead to etiologic treatment.

Phenotypic expression of a spectrum of Neurofibromatosis Type 1 (NF1) mutations identified through NGS and MLPA.

Neurofibromatosis Type 1 (NF1) is caused by mutations of the NF1 gene. The aim of this study was to identify the genetic causes underlying the disease, attempt possible phenotype/genotype correlations and add to the NF1 mutation spectrum. A screening protocol based on genomic DNA was established in 168 patients, encompassing sequencing of all coding exons and adjoining introns using a custom targeted next generation sequencing protocol and subsequent confirmation of findings with Sanger sequencing. MLPA was used to detect deletions/duplications and positive findings were confirmed by RNA analysis. All novel findings were evaluated according to ACMG Standards and guidelines for the interpretation of sequence variants with the aid of in-silico bioinformatic tools and family segregation analysis. A germline variant was identified in 145 patients (86%). In total 49 known and 70 novel variants in coding and non-coding regions were identified. Seven patients carried whole or partial gene deletions. NF1 patients, present with high phenotypic variability even in cases where the same germline disease causing variant has been identified. Our findings will contribute to a better knowledge of the genetic causes and the phenotypic expression related to the disease.

Rothmund-Thomson Syndrome: novel pathogenic mutations and frequencies of variants in the RECQL4 and USB1 (C16orf57) gene.

BACKGROUND: Poikiloderma is defined as a chronic skin condition presenting with a combination of punctate atrophy, areas of depigmentation, hyperpigmentation and telangiectasia. In a variety of hereditary syndromes such as Rothmund-Thomson syndrome (RTS), Clericuzio-type poikiloderma with neutropenia (PN) and Dyskeratosis Congenita (DC), poikiloderma occurs as one of the main symptoms. Here, we report on genotype and phenotype data of a cohort of 44 index patients with RTS or related genodermatoses. METHODS: DNA samples from 43 patients were screened for variants in the 21 exons of the RECQL4 gene using PCR, SSCP-PAGE analysis and/or Sanger sequencing. Patients with only one or no detectable mutation in the RECQL4 gene were additionally tested for variants in the 8 exons of the USB1 (C16orf57) gene by Sanger sequencing. The effect of novel variants was evaluated by phylogenic studies, single-nucleotide polymorphism (SNP) databases and in silico analyses. RESULTS: We identified 23 different RECQL4 mutations including 10 novel and one homozygous novel USB1 (C16orf57) mutation in a patient with PN. Moreover, we describe 31 RECQL4 and 8 USB1 sequence variants, four of them being novel intronic RECQL4 sequence changes that may have some deleterious effects on splicing mechanisms and need further evaluation by transcript analyses. CONCLUSION: The current study contributes to the improvement of genetic diagnostic strategies and interpretation in RTS and PN that is relevant in order to assess the patients' cancer risk, to avoid continuous and inconclusive clinical evaluations and to clarify the recurrence risk in the families. Additionally, it shows that the phenotype of more than 50% of the patients with suspected Rothmund-Thomson disease may be due to mutations in other genes raising the need for further extended genetic analyses.

Central precocious puberty in a boy with 22q13 deletion syndrome and NOTCH-1 gene duplication.

The 22q13 deletion syndrome or Phelan-McDermid syndrome is a neurodevelopmental disorder associated with developmental delay, hypotonia, delayed or absent speech, autistic-like behavior, normal to accelerated growth and dysmorphic faces. We report the occurrence of central precocious puberty in a boy diagnosed with Phelan-McDermid syndrome. At the age of 1 year, our patient presented with increased testicular volume for his age, bone age advancement and growth acceleration. Stimulated gonadotropin levels demonstrated a premature activation of the hypothalamic-pituitary-gonadal (HPG) axis. Central precocious puberty was treated with gonadotropin-releasing hormone (GnRH) analog. Molecular diagnosis with array-comparative genomic hybridization (CGH) revealed a major deletion of 5.8 Mb at the 22q13 chromosomal region and a 25 kb duplication at the 9q34.3 region that included the NOTCH-1 gene. On the background of 22q13 deletion syndrome and data from animals on the effect of abnormal NOTCH-1 gene expression on kisspeptin neuron formation, we discuss the probable role of Notch signaling in the premature activation of the HPG axis.

Lack of IL7Rα expression in T cells is a hallmark of T-cell immunodeficiency in Schimke immuno-osseous dysplasia (SIOD).

Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, fatal childhood disorder associated with skeletal dysplasia, renal dysfunction, and T-cell immunodeficiency. This disease is linked to biallelic loss-of-function mutations of the SMARCAL1 gene. Although recurrent infection, due to T-cell deficiency, is a leading cause of morbidity and mortality, the etiology of the T-cell immunodeficiency is unclear. Here, we demonstrate that the T cells of SIOD patients have undetectable levels of protein and mRNA for the IL-7 receptor alpha chain (IL7Rα) and are unresponsive to stimulation with IL-7, indicating a loss of functional receptor. No pathogenic mutations were detected in the exons of IL7R in these patients; however, CpG sites in the IL7R promoter were hypermethylated in SIOD T cells. We propose therefore that the lack of IL7Rα expression, associated with hypermethylation of the IL7R promoter, in T cells and possibly their earlier progenitors, restricts T-cell development in SIOD patients.

Erratum to: An interstitial deletion at 8q23.1-q24.12 associated with Langer-Giedion syndrome/ Trichorhinophalangeal syndrome (TRPS) type II and Cornelia de Lange syndrome 4.

[This corrects the article DOI: 10.1186/s13039-015-0169-9.].

An interstitial deletion at 8q23.1-q24.12 associated with Langer-Giedion syndrome/ Trichorhinophalangeal syndrome (TRPS) type II and Cornelia de Lange syndrome 4.

BACKGROUND: There are three distinct subtypes of Trichorhinophalangeal syndrome (TRPS); TRPS type I, TRPS type II and TRPS type III. Features common to all three subtypes include sparse, slowly growing scalp hair, laterally sparse eyebrows, a bulbous tip of the nose (pear-shaped), and protruding ears. Langer-Giedion syndrome (LGS) or TRPS type II is a contiguous gene syndrome on 8q24.1, involving loss of functional copies of the TRPS1 and EXT1 genes. We report a male patient that was referred to the Department of Medical Genetics due to hypotonia and dysmorphic facial features. RESULTS: Cytogenetic and array- Comparative Genomic Hybridization (aCGH) analysis revealed that the patient was a carrier of an interstitial deletion at 8q23.1-q24.12 of 12,5 Mb. Parental karyotype indicated that the father carried an apparently balanced insertion: 46, ΧΥ, der(10)ins(10;8)(q22;q23q24). CONCLUSIONS: This is the first report of an apparently balanced insertion including chromosomes 8 and 10 contributing to the etiology of LGS/ TRPS type II. Τimely diagnosis of parental balanced chromosomal rearrangements can reduce the risk of subsequent miscarriages as well as abnormal offspring.

A novel FOXL2 gene mutation and BMP15 variants in a woman with primary ovarian insufficiency and blepharophimosis-ptosis-epicanthus inversus syndrome.

OBJECTIVE: This study aims to search for mutations in relevant genes in a woman with primary ovarian insufficiency (POI) and blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). METHODS: This study reports on the case of a woman with POI, BPES, and autoimmune endocrine disorder. Bidirectional sequencing of the coding regions and intron/exon boundaries of FOXL2 and BMP15 genes and hormonal assays for the measurement of follicle-stimulating hormone, luteinizing hormone, estradiol, testosterone, Δ4-androstenedione, and dehydroepiandrosterone sulfate were employed. RESULTS: A novel de novo heterozygous deletion (p.K150Rfs*121) in the FOXL2 gene was identified to coexist with two BMP15 gene variants located in the same allele (c.-9C>G; p.N103S). CONCLUSIONS: The novel, de novo FOXL2 gene mutation (p.K150Rfs*121) expands the spectrum of molecular defects identified in women with BPES. Coexisting gene variants in POI-related genes, such as BMP15, may act synergistically and explain the observed phenotypic variability in women with BPES (ie, BPES with or without POI). The concept of digenic inheritance suggested herein has been previously introduced for other nosologies such as hypogonadotrophic hypogonadism. Endocrine autoimmunity might also contribute to the POI phenotype.

A novel large deletion of the ICR1 region including H19 and putative enhancer elements.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a rare pediatric overgrowth disorder with a variable clinical phenotype caused by deregulation affecting imprinted genes in the chromosomal region 11p15. Alterations of the imprinting control region 1 (ICR1) at the IGF2/H19 locus resulting in biallelic expression of IGF2 and biallelic silencing of H19 account for approximately 10% of patients with BWS. The majority of these patients have epimutations of the ICR1 without detectable DNA sequence changes. Only a few patients were found to have deletions. Most of these deletions are small affecting different parts of the ICR1 differentially methylated region (ICR1-DMR) removing target sequences for CTCF. Only a very few deletions reported so far include the H19 gene in addition to the CTCF binding sites. None of these deletions include IGF2. CASE PRESENTATION: A male patient was born with hypotonia, facial dysmorphisms and hypoglycemia suggestive of Beckwith-Wiedemann syndrome. Using methylation-specific (MS)-MLPA (Multiplex ligation-dependent probe amplification) we have identified a maternally inherited large deletion of the ICR1 region in a patient and his mother. The deletion results in a variable clinical expression with a classical BWS in the mother and a more severe presentation of BWS in her son. By genome-wide SNP array analysis the deletion was found to span ~100 kb genomic DNA including the ICR1DMR, H19, two adjacent non-imprinted genes and two of three predicted enhancer elements downstream to H19. Methylation analysis by deep bisulfite next generation sequencing revealed hypermethylation of the maternal allele at the IGF2 locus in both, mother and child, although IGF2 is not affected by the deletion. CONCLUSIONS: We here report on a novel large familial deletion of the ICR1 region in a BWS family. Due to the deletion of the ICR1-DMR CTCF binding cannot take place and the residual enhancer elements have access to the IGF2 promoters. The aberrant methylation (hypermethylation) of the maternal IGF2 allele in both affected family members may reflect the active state of the normally silenced maternal IGF2 copy and can be a consequence of the deletion. The deletion results in a variable clinical phenotype and expression.

Constitutional and somatic deletions of the Williams-Beuren syndrome critical region in non-Hodgkin lymphoma.

Here, we report and investigate the genomic alterations of two novel cases of Non-Hodgkin Lymphoma (NHL) in children with Williams-Beuren syndrome (WBS), a multisystem disorder caused by 7q11.23 hemizygous deletion. Additionally, we report the case of a child with NHL and a somatic 7q11.23 deletion. Although the WBS critical region has not yet been identified as a susceptibility locus in NHL, it harbors a number of genes involved in DNA repair. The high proportion of pediatric NHL reported in WBS is intriguing. Therefore, the role of haploinsufficiency of genes located at 7q11.23 in lymphomagenesis deserves to be investigated.

Diagnostic exome sequencing to elucidate the genetic basis of likely recessive disorders in consanguineous families.

Rare, atypical, and undiagnosed autosomal-recessive disorders frequently occur in the offspring of consanguineous couples. Current routine diagnostic genetic tests fail to establish a diagnosis in many cases. We employed exome sequencing to identify the underlying molecular defects in patients with unresolved but putatively autosomal-recessive disorders in consanguineous families and postulated that the pathogenic variants would reside within homozygous regions. Fifty consanguineous families participated in the study, with a wide spectrum of clinical phenotypes suggestive of autosomal-recessive inheritance, but with no definitive molecular diagnosis. DNA samples from the patient(s), unaffected sibling(s), and the parents were genotyped with a 720K SNP array. Exome sequencing and array CGH (comparative genomic hybridization) were then performed on one affected individual per family. High-confidence pathogenic variants were found in homozygosity in known disease-causing genes in 18 families (36%) (one by array CGH and 17 by exome sequencing), accounting for the clinical phenotype in whole or in part. In the remainder of the families, no causative variant in a known pathogenic gene was identified. Our study shows that exome sequencing, in addition to being a powerful diagnostic tool, promises to rapidly expand our knowledge of rare genetic Mendelian disorders and can be used to establish more detailed causative links between mutant genotypes and clinical phenotypes.

Clinical spectrum of females with HCCS mutation: from no clinical signs to a neonatal lethal form of the microphthalmia with linear skin defects (MLS) syndrome.

BACKGROUND: Segmental Xp22.2 monosomy or a heterozygous HCCS mutation is associated with the microphthalmia with linear skin defects (MLS) or MIDAS (microphthalmia, dermal aplasia, and sclerocornea) syndrome, an X-linked disorder with male lethality. HCCS encodes the holocytochrome c-type synthase involved in mitochondrial oxidative phosphorylation (OXPHOS) and programmed cell death. METHODS: We characterized the X-chromosomal abnormality encompassing HCCS or an intragenic mutation in this gene in six new female patients with an MLS phenotype by cytogenetic analysis, fluorescence in situ hybridization, sequencing, and quantitative real-time PCR. The X chromosome inactivation (XCI) pattern was determined and clinical data of the patients were reviewed. RESULTS: Two terminal Xp deletions of ≥ 11.2 Mb, two submicroscopic copy number losses, one of ~850 kb and one of ≥ 3 Mb, all covering HCCS, 1 nonsense, and one mosaic 2-bp deletion in HCCS are reported. All females had a completely (>98:2) or slightly skewed (82:18) XCI pattern. The most consistent clinical features were microphthalmia/anophthalmia and sclerocornea/corneal opacity in all patients and congenital linear skin defects in 4/6. Additional manifestations included various ocular anomalies, cardiac defects, brain imaging abnormalities, microcephaly, postnatal growth retardation, and facial dysmorphism. However, no obvious clinical sign was observed in three female carriers who were relatives of one patient. CONCLUSION: Our findings showed a wide phenotypic spectrum ranging from asymptomatic females with an HCCS mutation to patients with a neonatal lethal MLS form. Somatic mosaicism and the different ability of embryonic cells to cope with an OXPHOS defect and/or enhanced cell death upon HCCS deficiency likely underlie the great variability in phenotypes.

Array comparative genomic hybridization as a clinical diagnostic tool in syndromic and nonsyndromic congenital heart disease.

BACKGROUND: Congenital heart diseases (CHDs) are often associated with other congenital anomalies, dysmorphic features, and developmental delay, and only a few cases of chromosomal abnormalities are detected by conventional cytogenetic techniques. The microarray comparative genomic hybridization (CGH) analysis allows the identification of submicroscopic genomic rearrangements. METHODS: During the past 3 y, 55 of 330 patients referred for array CGH had CHD of unknown etiology plus at least one additional indication of abnormal chromosomal phenotype. High-resolution 1 × 244 K or 4 × 180 K Agilent arrays were used in this study (average resolution 7-13 kb). RESULTS: Copy-number variations were detected in 37 of 55 patients, and in 29 of 37 patients there were genes that have been associated with CHD. All 37 patients had at least one additional phenotypic abnormality: 30 of 37 had one or more other congenital anomalies, 23 of 37 had dysmorphic features, 16 of 37 had intellectual disability, 13 of 37 had abnormal magnetic resonance imaging, 10 of 37 had hypotonia, and 7 of 37 had seizures. In 9 of 55 patients, unexpected genomic rearrangements in relation to their phenotype were identified. CONCLUSION: In patients with CHD and at least one additional indication of abnormal chromosomal phenotype, array CGH analysis could detect possible submicroscopic chromosomal abnormalities and provide proper genetic counseling.

Array-CGH revealed one of the smallest 16q21q22.1 microdeletions in a female patient with psychomotor retardation.

A 28-month-old girl with dysmorphic craniofacial features, microcephaly, hypotonia, psychomotor retardation, failure to thrive and gastrointestinal problems was referred for clinical evaluation. Array-CGH analysis revealed one of the smallest de novo microdeletions on chromosome 16q21q22.1, 2.03 Mb in size. Advanced molecular analysis contributes to more precise genotype-phenotype correlation and accurate definition of the breakpoints in the deleted/duplicated regions.

COMT and MTHFR polymorphisms interaction on cognition in schizophrenia: an exploratory study.

The investigation of the catechol-O-methyltransferase (COMT-[rs4680]) and methylenetetrahydrofolate reductase (MTHFR-[rs1801133]) polymorphisms' interaction might shed light into the pathogenetic mechanisms of the cognitive dysfunction in schizophrenia. In an exploratory study, we hypothesized that the MTHFR 677T allele which has been related to a hypoactive MTHFR enzyme would augment the unfavorable effects of COMT Val158 homozygosity which has been associated with COMT enzyme hyperfunction. 90 schizophrenia patients and 55 healthy volunteers were assessed on psychomotor speed, pattern and spatial recognition memory (SRM), spatial working memory (SWM), attentional flexibility and planning (Stockings of Cambridge-SOC). IQ scores in a random subgroup of patients were also measured. A significant COMT×MTHFR interaction on SWM (p=0.048) and planning (p=0.026) was revealed in both groups. Among COMT-Val/Val participants, MTHFR-C/C made more SWM errors (p=0.033) and solved fewer SOC problems (p=0.025) than MTHFR-T carriers. In patients, there was a significant COMT×MTHFR interaction on full scale IQ (p=0.035): among COMT-Met carriers, MTHFR-T carriers performed significantly worse than MTHFR-C/C (p=0.021), which was driven by a COMT×MTHFR interaction involving performance IQ (p=0.047). In conclusion, COMT and MTHFR polymorphisms interacted on cognition, suggesting that the MTHFR enzyme activity might moderate the effects of the COMT enzyme. In contrast to our initial hypothesis, the MTHFR T-allele attenuated the cognitive effects of COMT Val homozygosity. In this preliminary study, we propose that dopaminergic and intracellular methylation mechanisms could interact on cognitive deficits in schizophrenia.

Reduced elastogenesis: a clue to the arteriosclerosis and emphysematous changes in Schimke immuno-osseous dysplasia?

BACKGROUND: Arteriosclerosis and emphysema develop in individuals with Schimke immuno-osseous dysplasia (SIOD), a multisystem disorder caused by biallelic mutations in SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1). However, the mechanism by which the vascular and pulmonary disease arises in SIOD remains unknown. METHODS: We reviewed the records of 65 patients with SMARCAL1 mutations. Molecular and immunohistochemical analyses were conducted on autopsy tissue from 4 SIOD patients. RESULTS: Thirty-two of 63 patients had signs of arteriosclerosis and 3 of 51 had signs of emphysema. The arteriosclerosis was characterized by intimal and medial hyperplasia, smooth muscle cell hyperplasia and fragmented and disorganized elastin fibers, and the pulmonary disease was characterized by panlobular enlargement of air spaces. Consistent with a cell autonomous disorder, SMARCAL1 was expressed in arterial and lung tissue, and both the aorta and lung of SIOD patients had reduced expression of elastin and alterations in the expression of regulators of elastin gene expression. CONCLUSIONS: This first comprehensive study of the vascular and pulmonary complications of SIOD shows that these commonly cause morbidity and mortality and might arise from impaired elastogenesis. Additionally, the effect of SMARCAL1 deficiency on elastin expression provides a model for understanding other features of SIOD.