ITCH deficiency clinical phenotype expansion and mitochondrial dysfunction.

Autoimmune Disease, Multisystem, with Facial Dysmorphism (ADMFD) is an autosomal recessive disorder due to pathogenic variants in the ITCH gene. It is characterized by failure to thrive, dysmorphic facial features, developmental delay, and systemic autoimmunity that can manifest variably with autoimmune hepatitis, thyroiditis, and enteropathy, among other organ manifestations. It was originally described in 10 consanguineous Old Order Amish patients, and more recently in two patients of White British and Black German ethnicities. While the role of ITCH protein in apoptosis and inflammation has previously been characterized, a defect in cellular bioenergetics has not yet been reported in ITCH deficiency. Here we present a Caucasian female originally evaluated for possible mitochondrial respiratory chain deficiency, who ultimately was found to have two novel variants in ITCH with absence of ITCH protein in patient derived fibroblasts. Clinical studies of patient muscle showed mitochondrial DNA copy number of 57% compared to controls. Functional studies in skin fibroblasts revealed decreased activity of mitochondrial fatty acid oxidation and oxidative phosphorylation, and decreased overall ATP production. Our findings confirm mitochondrial energy dysfunction in a patient with ITCH deficiency offering the opportunity to assess alternative therapeutic options.

Deletion of conserved non-coding sequences downstream from NKX2-1: A novel disease-causing mechanism for benign hereditary chorea.

BACKGROUND: Benign hereditary chorea (BHC) is an autosomal dominant disorder characterized by early-onset non-progressive involuntary movements. Although NKX2-1 mutations or deletions are the cause of BHC, some BHC families do not have pathogenic alterations in the NKX2-1 gene, indicating that mutations of non-coding regulatory elements of NKX2-1 may also play a role. METHODS AND RESULTS: By using whole-genome microarray analysis, we identified a 117 Kb founder deletion in three apparently unrelated BHC families that were negative for NKX2-1 sequence variants. Targeted next generation sequencing analysis confirmed the deletion and showed that it was part of a complex local genomic rearrangement. In addition, we also detected a 648 Kb de novo deletion in an isolated BHC case. Both deletions are located downstream from NKX2-1 on chromosome 14q13.2-q13.3 and share a 33 Kb smallest region of overlap with six previously reported cases. This region has no gene but contains multiple evolutionarily highly conserved non-coding sequences. CONCLUSION: We propose that the deletion of potential regulatory elements necessary for NKX2-1 expression in this critical region is responsible for BHC phenotype in these patients, and this is a novel disease-causing mechanism for BHC.

Disruption of PHF21A causes syndromic intellectual disability with craniofacial anomalies, epilepsy, hypotonia, and neurobehavioral problems including autism.

Background: PHF21A has been associated with intellectual disability and craniofacial anomalies based on its deletion in the Potocki-Shaffer syndrome region at 11p11.2 and its disruption in three patients with balanced translocations. In addition, three patients with de novo truncating mutations in PHF21A were reported recently. Here, we analyze genomic data from seven unrelated individuals with mutations in PHF21A and provide detailed clinical descriptions, further expanding the phenotype associated with PHF21A haploinsufficiency. Methods: Diagnostic trio whole exome sequencing, Sanger sequencing, use of GeneMatcher, targeted gene panel sequencing, and MiSeq sequencing techniques were used to identify and confirm variants. RT-qPCR was used to measure the normal expression pattern of PHF21A in multiple human tissues including 13 different brain tissues. Protein-DNA modeling was performed to substantiate the pathogenicity of the missense mutation. Results: We have identified seven heterozygous coding mutations, among which six are de novo (not maternal in one). Mutations include four frameshifts, one nonsense mutation in two patients, and one heterozygous missense mutation in the AT Hook domain, predicted to be deleterious and likely to cause loss of PHF21A function. We also found a new C-terminal domain composed of an intrinsically disordered region. This domain is truncated in six patients and thus likely to play an important role in the function of PHF21A, suggesting that haploinsufficiency is the likely underlying mechanism in the phenotype of seven patients. Our results extend the phenotypic spectrum of PHF21A mutations by adding autism spectrum disorder, epilepsy, hypotonia, and neurobehavioral problems. Furthermore, PHF21A is highly expressed in the human fetal brain, which is consistent with the neurodevelopmental phenotype. Conclusion: Deleterious nonsense, frameshift, and missense mutations disrupting the AT Hook domain and/or an intrinsically disordered region in PHF21A were found to be associated with autism spectrum disorder, epilepsy, hypotonia, neurobehavioral problems, tapering fingers, clinodactyly, and syndactyly, in addition to intellectual disability and craniofacial anomalies. This suggests that PHF21A is involved in autism spectrum disorder and intellectual disability, and its haploinsufficiency causes a diverse neurological phenotype.

Clinical exome sequencing reveals locus heterogeneity and phenotypic variability of cohesinopathies.

PURPOSE: Defects in the cohesin pathway are associated with cohesinopathies, notably Cornelia de Lange syndrome (CdLS). We aimed to delineate pathogenic variants in known and candidate cohesinopathy genes from a clinical exome perspective. METHODS: We retrospectively studied patients referred for clinical exome sequencing (CES, N = 10,698). Patients with causative variants in novel or recently described cohesinopathy genes were enrolled for phenotypic characterization. RESULTS: Pathogenic or likely pathogenic single-nucleotide and insertion/deletion variants (SNVs/indels) were identified in established disease genes including NIPBL (N = 5), SMC1A (N = 14), SMC3 (N = 4), RAD21 (N = 2), and HDAC8 (N = 8). The phenotypes in this genetically defined cohort skew towards the mild end of CdLS spectrum as compared with phenotype-driven cohorts. Candidate or recently reported cohesinopathy genes were supported by de novo SNVs/indels in STAG1 (N = 3), STAG2 (N = 5), PDS5A (N = 1), and WAPL (N = 1), and one inherited SNV in PDS5A. We also identified copy-number deletions affecting STAG1 (two de novo, one of unknown inheritance) and STAG2 (one of unknown inheritance). Patients with STAG1 and STAG2 variants presented with overlapping features yet without characteristic facial features of CdLS. CONCLUSION: CES effectively identified disease-causing alleles at the mild end of the cohensinopathy spectrum and enabled characterization of candidate disease genes.

De novo apparent loss-of-function mutations in PRR12 in three patients with intellectual disability and iris abnormalities.

PRR12 encodes a proline-rich protein nuclear factor suspected to be involved in neural development. Its nuclear expression in fetal brains and in the vision system supports its role in brain and eye development more specifically. However, its function and potential role in human disease has not been determined. Recently, a de novo t(10;19) (q22.3;q13.33) translocation disrupting the PRR12 gene was detected in a girl with intellectual disability and neuropsychiatric alterations. Here we report on three unrelated patients with heterozygous de novo apparent loss-of-function mutations in PRR12 detected by clinical whole exome sequencing: c.1918G>T (p.Glu640*), c.4502_4505delTGCC (p.Leu1501Argfs*146) and c.903_909dup (p.Pro304Thrfs*46). All three patients had global developmental delay, intellectual disability, eye and vision abnormalities, dysmorphic features, and neuropsychiatric problems. Eye abnormalities were consistent among the three patients and consisted of stellate iris pattern and iris coloboma. Additional variable clinical features included hypotonia, skeletal abnormalities, sleeping problems, and behavioral issues such as autism and anxiety. In summary, we propose that haploinsufficiency of PRR12 is associated with this novel multisystem neurodevelopmental disorder.

Kaufman oculocerebrofacial syndrome: Novel UBE3B mutations and clinical features in four unrelated patients.

The "blepharophimosis-mental retardation" syndromes (BMRS) consist of a group of clinically and genetically heterogeneous congenital malformation syndromes, where short palpebral fissures and intellectual disability associate with a distinct set of other morphological features. Kaufman oculocerebrofacial syndrome represents a rare and recently reevaluated entity within the BMR syndromes and is caused by biallelic mutations of UBE3B. Affected individuals typically show microcephaly, impaired somatic growth, gastrointestinal and genitourinary problems, ectodermal anomalies and a characteristic face with short, upslanted palpebral fissures, depressed nasal bridge. and anteverted nares. Here we present four patients with five novel UBE3B mutations and propose the inclusion of clinical features to the characteristics of Kaufman oculocerebrofacial syndrome, including prominence of the cheeks and limb anomalies.

Phenotypic and molecular characterisation of CDK13-related congenital heart defects, dysmorphic facial features and intellectual developmental disorders.

BACKGROUND: De novo missense variants in CDK13 have been described as the cause of syndromic congenital heart defects in seven individuals ascertained from a large congenital cardiovascular malformations cohort. We aimed to further define the phenotypic and molecular spectrum of this newly described disorder. METHODS: To minimise ascertainment bias, we recruited nine additional individuals with CDK13 pathogenic variants from clinical and research exome laboratory sequencing cohorts. Each individual underwent dysmorphology exam and comprehensive medical history review. RESULTS: We demonstrate greater than expected phenotypic heterogeneity, including 33% (3/9) of individuals without structural heart disease on echocardiogram. There was a high penetrance for a unique constellation of facial dysmorphism and global developmental delay, as well as less frequently seen renal and sacral anomalies. Two individuals had novel CDK13 variants (p.Asn842Asp, p.Lys734Glu), while the remaining seven unrelated individuals had a recurrent, previously published p.Asn842Ser variant. Summary of all variants published to date demonstrates apparent restriction of pathogenic variants to the protein kinase domain with clustering in the ATP and magnesium binding sites. CONCLUSIONS: Here we provide detailed phenotypic and molecular characterisation of individuals with pathogenic variants in CDK13 and propose management guidelines based upon the estimated prevalence of anomalies identified.

Germline mutations in ABL1 cause an autosomal dominant syndrome characterized by congenital heart defects and skeletal malformations.

ABL1 is a proto-oncogene well known as part of the fusion gene BCR-ABL1 in the Philadelphia chromosome of leukemia cancer cells. Inherited germline ABL1 changes have not been associated with genetic disorders. Here we report ABL1 germline variants cosegregating with an autosomal dominant disorder characterized by congenital heart disease, skeletal abnormalities, and failure to thrive. The variant c.734A>G (p.Tyr245Cys) was found to occur de novo or cosegregate with disease in five individuals (families 1-3). Additionally, a de novo c.1066G>A (p.Ala356Thr) variant was identified in a sixth individual (family 4). We overexpressed the mutant constructs in HEK 293T cells and observed increased tyrosine phosphorylation, suggesting increased ABL1 kinase activities associated with both the p.Tyr245Cys and p.Ala356Thr substitutions. Our clinical and experimental findings, together with previously reported teratogenic effects of selective BCR-ABL inhibitors in humans and developmental defects in Abl1 knockout mice, suggest that ABL1 has an important role during organismal development.

GRIN1 mutation associated with intellectual disability alters NMDA receptor trafficking and function.

N-methyl-d-aspartate receptors (NMDARs) play important roles in brain development and neurological disease. We report two individuals with similar dominant de novo GRIN1 mutations (c.1858 G>A and c.1858 G>C; both p.G620R). Both individuals presented at birth with developmental delay and hypotonia associated with behavioral abnormalities and stereotypical movements. Recombinant NMDARs containing the mutant GluN1-G620R together with either GluN2A or GluN2B were evaluated for changes in their trafficking to the plasma membrane and their electrophysiological properties. GluN1-G620R/GluN2A complexes showed a mild reduction in trafficking, a ~2-fold decrease in glutamate and glycine potency, a strong decrease in sensitivity to Mg2+ block, and a significant reduction of current responses to a maximal effective concentration of agonists. GluN1-G620R/GluN2B complexes showed significantly reduced delivery of protein to the cell surface associated with similarly altered electrophysiology. These results indicate these individuals may have suffered neurodevelopmental deficits as a result of the decreased presence of GluN1-G620R/GluN2B complexes on the neuronal surface during embryonic brain development and reduced current responses of GluN1-G620R-containing NMDARs after birth. These cases emphasize the importance of comprehensive functional characterization of de novo mutations and illustrates how a combination of several distinct features of NMDAR expression, trafficking and function can be present and influence phenotype.

De Novo Truncating Variants in SON Cause Intellectual Disability, Congenital Malformations, and Failure to Thrive.

SON is a key component of the spliceosomal complex and a critical mediator of constitutive and alternative splicing. Additionally, SON has been shown to influence cell-cycle progression, genomic integrity, and maintenance of pluripotency in stem cell populations. The clear functional relevance of SON in coordinating essential cellular processes and its presence in diverse human tissues suggests that intact SON might be crucial for normal growth and development. However, the phenotypic effects of deleterious germline variants in SON have not been clearly defined. Herein, we describe seven unrelated individuals with de novo variants in SON and propose that deleterious variants in SON are associated with a severe multisystem disorder characterized by developmental delay, persistent feeding difficulties, and congenital malformations, including brain anomalies.

Respiratory motile cilia dysfunction in a patient with cranioectodermal dysplasia.

Ciliopathies such as cranioectodermal dysplasia, Sensenbrenner syndrome, short-rib polydactyly, and Jeune syndrome are associated with respiratory complications arising from rib cage dysplasia. While such ciliopathies have been demonstrated to involve primary cilia defects, we show motile cilia dysfunction in the airway of a patient diagnosed with cranioectodermal dysplasia. While this patient had mild thoracic dystrophy not requiring surgical treatment, there was nevertheless newborn respiratory distress, restrictive airway disease with possible obstructive airway involvement, repeated respiratory infections, and atelectasis. High-resolution videomicroscopy of nasal epithelial biopsy showed immotile/dyskinetic cilia and nasal nitric oxide was reduced, both of which are characteristics of primary ciliary dyskinesia, a sinopulmonary disease associated with mucociliary clearance defects due to motile cilia dysfunction in the airway. Exome sequencing analysis of this patient identified compound heterozygous mutations in WDR35, but no mutations in any of the 30 known primary ciliary dyskinesia genes or other cilia-related genes. Given that WDR35 is only known to be required for primary cilia function, we carried out WDR35 siRNA knockdown in human respiratory epithelia to assess the role of WDR35 in motile cilia function. This showed WDR35 deficiency disrupted ciliogenesis in the airway, indicating WDR35 is also required for formation of motile cilia. Together, these findings suggest patients with WDR35 mutations have an airway mucociliary clearance defect masked by their restrictive airway disease.

Craniofacial abnormalities and developmental delay in two families with overlapping 22q12.1 microdeletions involving the MN1 gene.

Deletions spanning the MN1 gene (22q12.1) have recently been proposed as playing a role in craniofacial abnormalities that include cleft palate, as mouse studies have demonstrated that Mn1 haploinsufficiency results in skull abnormalities and secondary cleft palate. We report on four patients (two families) with craniofacial abnormalities and intellectual disability with overlapping microdeletions that span the MN1 gene. Comparative genomic hybridization microarray analysis revealed a 2.76 Mb deletion in the 22q12.1 region, in three family members (Family 1), that contains the MN1 gene. In addition, a complex 22q12 rearrangement, including a 1.61 Mb deletion containing the MN1 gene and a 2.28 Mb deletion encompassing the NF2 gene, has been identified in another unrelated patient (Family 2). Based upon genotype-phenotype correlation among our patients and those previously reported with overlapping 22q12 deletions, we identified a 560 kb critical region containing the MN1 gene that is implicated in human cleft palate formation. Importantly, NF2 was also found within the 22q12 deletion region in several patients which enabled specific clinical management for neurofibromatosis 2.

Mutations in PURA cause profound neonatal hypotonia, seizures, and encephalopathy in 5q31.3 microdeletion syndrome.

5q31.3 microdeletion syndrome is characterized by neonatal hypotonia, encephalopathy with or without epilepsy, and severe developmental delay, and the minimal critical deletion interval harbors three genes. We describe 11 individuals with clinical features of 5q31.3 microdeletion syndrome and de novo mutations in PURA, encoding transcriptional activator protein Pur-α, within the critical region. These data implicate causative PURA mutations responsible for the severe neurological phenotypes observed in this syndrome.

Co-existence of 9p deletion and Silver-Russell syndromes in a patient with maternally inherited cryptic complex chromosome rearrangement involving chromosomes 4, 9, and 11.

We report a patient with a maternally inherited unbalanced complex chromosomal rearrangement (CCR) involving chromosomes 4, 9, and 11 detected by microarray comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). This patient presents with clinical features of 9p deletion syndrome and Silver-Russell syndrome (SRS). Chromosome analysis performed in 2000 showed what appeared to be a simple terminal deletion of chromosome 9p22.1. aCGH performed in 2010 revealed a 1.63 Mb duplication at 4q28.3, a 15.48 Mb deletion at 9p24.3p22.3, and a 1.95 Mb duplication at 11p15.5. FISH analysis revealed a derivative chromosome 9 resulting from an unbalanced translocation between chromosomes 9 and 11, a chromosome 4 fragment inserted near the breakpoint of the translocation. The 4q28.3 duplication does not contain any currently known genes. The 9p24.3p22.3 deletion region contains 36 OMIM genes including a 3.5 Mb critical region for the 9p-phenotype. The 11p15.5 duplication contains 49 OMIM genes including H19 and IGF2. Maternal aCGH was normal. However, maternal chromosomal and FISH analyses revealed an apparently balanced CCR involving chromosomes 4, 9, and 11. To the best of our knowledge, this is the first report of a patient with maternally inherited trans-duplication of the entire imprinting control region 1 (ICR1) among the 11p15.5 duplications reported in SRS patients. This report supports the hypothesis that the trans-duplication of the maternal copy of ICR1 alone is sufficient for the clinical manifestation of SRS and demonstrates the usefulness of combining aCGH with karyotyping and FISH for detecting cryptic genomic imbalances.

Phenotypic heterogeneity of genomic disorders and rare copy-number variants.

BACKGROUND: Some copy-number variants are associated with genomic disorders with extreme phenotypic heterogeneity. The cause of this variation is unknown, which presents challenges in genetic diagnosis, counseling, and management. METHODS: We analyzed the genomes of 2312 children known to carry a copy-number variant associated with intellectual disability and congenital abnormalities, using array comparative genomic hybridization. RESULTS: Among the affected children, 10.1% carried a second large copy-number variant in addition to the primary genetic lesion. We identified seven genomic disorders, each defined by a specific copy-number variant, in which the affected children were more likely to carry multiple copy-number variants than were controls. We found that syndromic disorders could be distinguished from those with extreme phenotypic heterogeneity on the basis of the total number of copy-number variants and whether the variants are inherited or de novo. Children who carried two large copy-number variants of unknown clinical significance were eight times as likely to have developmental delay as were controls (odds ratio, 8.16; 95% confidence interval, 5.33 to 13.07; P=2.11×10(-38)). Among affected children, inherited copy-number variants tended to co-occur with a second-site large copy-number variant (Spearman correlation coefficient, 0.66; P<0.001). Boys were more likely than girls to have disorders of phenotypic heterogeneity (P<0.001), and mothers were more likely than fathers to transmit second-site copy-number variants to their offspring (P=0.02). CONCLUSIONS: Multiple, large copy-number variants, including those of unknown pathogenic significance, compound to result in a severe clinical presentation, and secondary copy-number variants are preferentially transmitted from maternal carriers. (Funded by the Simons Foundation Autism Research Initiative and the National Institutes of Health.).

Subtelomeric deletion of chromosome 10p15.3: clinical findings and molecular cytogenetic characterization.

We describe 19 unrelated individuals with submicroscopic deletions involving 10p15.3 characterized by chromosomal microarray (CMA). Interestingly, to our knowledge, only two individuals with isolated, submicroscopic 10p15.3 deletion have been reported to date; however, only limited clinical information is available for these probands and the deleted region has not been molecularly mapped. Comprehensive clinical history was obtained for 12 of the 19 individuals described in this study. Common features among these 12 individuals include: cognitive/behavioral/developmental differences (11/11), speech delay/language disorder (10/10), motor delay (10/10), craniofacial dysmorphism (9/12), hypotonia (7/11), brain anomalies (4/6) and seizures (3/7). Parental studies were performed for nine of the 19 individuals; the 10p15.3 deletion was de novo in seven of the probands, not maternally inherited in one proband and inherited from an apparently affected mother in one proband. Molecular mapping of the 19 individuals reported in this study has identified two genes, ZMYND11 (OMIM 608668) and DIP2C (OMIM 611380; UCSC Genome Browser), mapping within 10p15.3 which are most commonly deleted. Although no single gene has been identified which is deleted in all 19 individuals studied, the deleted region in all but one individual includes ZMYND11 and the deleted region in all but one other individual includes DIP2C. There is not a clearly identifiable phenotypic difference between these two individuals and the size of the deleted region does not generally predict clinical features. Little is currently known about these genes complicating a direct genotype/phenotype correlation at this time. These data however, suggest that ZMYND11 and/or DIP2C haploinsufficiency contributes to the clinical features associated with 10p15 deletions in probands described in this study.

High-resolution array CGH defines critical regions and candidate genes for microcephaly, abnormalities of the corpus callosum, and seizure phenotypes in patients with microdeletions of 1q43q44.

Microdeletions of 1q43q44 result in a recognizable clinical disorder characterized by moderate to severe intellectual disability (ID) with limited or no expressive speech, characteristic facial features, hand and foot anomalies, microcephaly (MIC), abnormalities (agenesis/hypogenesis) of the corpus callosum (ACC), and seizures (SZR). Critical regions have been proposed for some of the more prominent features of this disorder such as MIC and ACC, yet conflicting data have prevented precise determination of the causative genes. In this study, the largest of pure interstitial and terminal deletions of 1q43q44 to date, we characterized 22 individuals by high-resolution oligonucleotide microarray-based comparative genomic hybridization. We propose critical regions and candidate genes for the MIC, ACC, and SZR phenotypes associated with this microdeletion syndrome. Three cases with MIC had small overlapping or intragenic deletions of AKT3, an isoform of the protein kinase B family. The deletion of only AKT3 in two cases implicates haploinsufficiency of this gene in the MIC phenotype. Likewise, based on the smallest region of overlap among the affected individuals, we suggest a critical region for ACC that contains ZNF238, a transcriptional and chromatin regulator highly expressed in the developing and adult brain. Finally, we describe a critical region for the SZR phenotype which contains three genes (FAM36A, C1ORF199, and HNRNPU). Although ~90% of cases in this study and in the literature fit these proposed models, the existence of phenotypic variability suggests other mechanisms such as variable expressivity, incomplete penetrance, position effects, or multigenic factors could account for additional complexity in some cases.

Erratum: GTP-cyclohydrolase deficiency responsive to sapropterin and 5-HTP supplementation: relief of treatment-refractory depression and suicidal behaviour.

[This corrects the article DOI: 10.1136/bcr.03.2011.3927.].

GTP-cyclohydrolase deficiency responsive to sapropterin and 5-HTP supplementation: relief of treatment-refractory depression and suicidal behaviour.

The authors describe a new variant of guanosine triphosphate (GTP)- cyclohydrolase deficiency in a young man with severe and disabling major depressive disorder with multiple near-lethal suicide attempts. His cerebrospinal fluid levels showed that the concentration of tetrahydrobiopterin (BH4), neopterin, 5-hydroxyindoleacetic acid and homovanillic acid were below the reference range, suggesting a defect in the pterin biosynthetic pathway and in synthesis of dopamine and serotonin indicative of GTP-cyclohydrolase deficiency. Patient was started on sapropterin, a BH4 replacement protein, for the defect in the above pathway. In addition, the authors started 5-hydroxytryptophan titrated to 400 mg orally twice daily with concomittant carbidopa 37.5 mg orally four times a day, and he responded with remission of suicidal ideation and significant improvement in depression and function.