Novel loss-of-function variants expand ABCC9-related intellectual disability and myopathy syndrome.

Loss-of-function mutation of ABCC9, the gene encoding the SUR2 subunit of ATP sensitive-potassium (KATP) channels, was recently associated with autosomal recessive ABCC9-related intellectual disability and myopathy syndrome (AIMS). Here we identify nine additional subjects, from seven unrelated families, harbouring different homozygous loss-of-function variants in ABCC9 and presenting with a conserved range of clinical features. All variants are predicted to result in severe truncations or in-frame deletions within SUR2, leading to the generation of non-functional SUR2-dependent KATP channels. Affected individuals show psychomotor delay and intellectual disability of variable severity, microcephaly, corpus callosum and white matter abnormalities, seizures, spasticity, short stature, muscle fatigability and weakness. Heterozygous parents do not show any conserved clinical pathology but report multiple incidences of intra-uterine fetal death, which were also observed in an eighth family included in this study. In vivo studies of abcc9 loss-of-function in zebrafish revealed an exacerbated motor response to pentylenetetrazole, a pro-convulsive drug, consistent with impaired neurodevelopment associated with an increased seizure susceptibility. Our findings define an ABCC9 loss-of-function-related phenotype, expanding the genotypic and phenotypic spectrum of AIMS and reveal novel human pathologies arising from KATP channel dysfunction.

Loss of CBY1 results in a ciliopathy characterized by features of Joubert syndrome.

Ciliopathies are clinically and genetically heterogeneous diseases. We studied three patients from two independent families presenting with features of Joubert syndrome: abnormal breathing pattern during infancy, developmental delay/intellectual disability, cerebellar ataxia, molar tooth sign on magnetic resonance imaging scans, and polydactyly. We identified biallelic loss-of-function (LOF) variants in CBY1, segregating with the clinical features of Joubert syndrome in the families. CBY1 localizes to the distal end of the mother centriole, contributing to the formation and function of cilia. In accordance with the clinical and mutational findings in the affected individuals, we demonstrated that depletion of Cby1 in zebrafish causes ciliopathy-related phenotypes. Levels of CBY1 transcript were found reduced in the patients compared with controls, suggesting degradation of the mutated transcript through nonsense-mediated messenger RNA decay. Accordingly, we could detect CBY1 protein in fibroblasts from controls, but not from patients by immunofluorescence. Furthermore, we observed reduced ability to ciliate, increased ciliary length, and reduced levels of the ciliary proteins AHI1 and ARL13B in patient fibroblasts. Our data show that CBY1 LOF-variants cause a ciliopathy with features of Joubert syndrome.

A novel type of rhizomelic chondrodysplasia punctata, RCDP5, is caused by loss of the PEX5 long isoform.

Import of peroxisomal matrix proteins, crucial for peroxisome biogenesis, is mediated by the cytosolic receptors PEX5 and PEX7 that recognize proteins carrying peroxisomal targeting signals 1 or 2 (PTS1 or PTS2), respectively. Mutations in PEX5 or 12 other PEX genes cause peroxisome biogenesis disorders, collectively named the Zellweger spectrum disorders (ZSDs), whereas mutations in PEX7 cause rhizomelic chondrodysplasia punctata type 1 (RCDP1). Three additional RCDP types, RCDP2-3-4, are caused, respectively, by mutations in GNPAT, AGPS and FAR1, encoding enzymes involved in plasmalogen biosynthesis. Here we report a fifth type of RCDP (RCDP5) caused by a novel mutation in PEX5. In four patients with RCDP from two independent families, we identified a homozygous frame shift mutation c.722dupA (p.Val242Glyfs(∗)33) in PEX5 (GenBank: NM_001131023.1). PEX5 encodes two isoforms, PEX5L and PEX5S, and we show that the c.722dupA mutation, located in the PEX5L-specific exon 9, results in loss of PEX5L only. Both PEX5 isoforms recognize PTS1-tagged proteins, but PEX5L is also a co-receptor for PTS2-tagged proteins. Previous patients with PEX5 mutations had ZSD, mainly due to deficient import of PTS1-tagged proteins. Similarly to mutations in PEX7, loss of PEX5L results in deficient import of PTS2-tagged proteins only, thus causing RCDP instead of ZSD. We demonstrate that PEX5L expression restores the import of PTS2-tagged proteins in patient fibroblasts. Due to the biochemical overlap between RCDP1 and RCDP5, sequencing of PEX7 and exon 9 in PEX5 should be performed in patients with a selective defect in the import of PTS2-tagged proteins.

Qualitative and quantitative analysis of FBN1 mRNA from 16 patients with Marfan Syndrome.

BACKGROUND: Pathogenic mutations in FBN1, encoding the glycoprotein, fibrillin-1, cause Marfan syndrome (MFS) and related connective tissue disorders. In the present study, qualitative and quantitative effects of 16 mutations, identified in FBN1 in MFS patients with systematically described phenotypes, were investigated in vitro. METHODS: Qualitative analysis was performed with reverse transcription-PCR (RT-PCR) and gel electrophoresis, and quantitative analysis to determine the FBN1 mRNA levels in fibroblasts from the 16 patients with MFS was performed with real-time PCR. RESULTS: Qualitative analysis documented that the mutations c.4817-2delA and c.A4925G led to aberrant FBN1 mRNA splicing leading to in frame deletion of exon 39 and in exon 39, respectively. No difference in the mean FBN1 mRNA level was observed between the entire group of cases and controls, nor between the group of patients with missense mutations and controls. The mean expression levels associated with premature termination codon (PTC) and splice site mutations were significantly lower than the levels in patients with missense mutations. A high level of FBN1 mRNA in the patient with the missense mutation c.G2447T did not segregate with the mutation in three of his first degree relatives. No association was indicated between the FBN1 transcript level and specific phenotypic manifestations. CONCLUSIONS: Abnormal FBN1 transcripts were indicated in fibroblasts from patients with the splice site mutation c.4817-2delA and the missense mutation c.A4925G. While the mean FBN1 mRNA expression level in fibroblasts from patients with splice site and PTC mutations were lower than the mean level in patients with missense mutations and controls, inter-individual variability was high. The observation that high level of FBN1 mRNA in the patient with the missense mutation c.G2447T did not segregate with the mutation in the family suggests that variable expression of the normal FBN1 allele may contribute to explain the variability in FBN1 mRNA level.

Kaufman oculocerebrofacial syndrome in sisters with novel compound heterozygous mutation in UBE3B.

A pair of sisters was ascertained for multiple congenital defects, including marked craniofacial dysmorphisms with blepharophimosis, and severe psychomotor delay. Two novel compound heterozygous mutations in UBE3B were identified in both the sisters by exome sequencing. These mutations include c.1A>G, which predicts p.Met1?, and a c.1773delC variant, predicted to cause a frameshift at p.Phe591fs. UBE3B encodes a widely expressed protein ubiquitin ligase E3B, which, when mutated in both alleles, causes Kaufman oculocerebrofacial syndrome. We report on the thorough clinical examination of the patients and review the state of art knowledge of this disorder.

Hyperphagia, mild developmental delay but apparently no structural brain anomalies in a boy without SOX3 expression.

The transcription factor SOX3 is widely expressed in early vertebrate brain development. In humans, duplication of SOX3 and polyalanine expansions at its C-terminus may cause intellectual disability and hypopituitarism. Sox3 knock-out mice show a variable phenotype including structural and functional anomalies affecting the branchial arches and midline cerebral structures such as the optic chiasm and the hypothalamo-pituitary axis. SOX3 is claimed to be required in normal brain development and function in mice and humans, as well as in pituitary and craniofacial development. We report on an 8-year-old boy with a 2.1 Mb deletion in Xq27.1q27.2, which was found to be inherited from his healthy mother. To our knowledge, this is the smallest deletion including the entire SOX3 gene in a male reported to date. He is mildly intellectually disabled with language delay, dysarthria, behavior problems, minor facial anomalies, and hyperphagia. Hormone levels including growth, adrenocorticotropic and thyroid stimulating hormones are normal. Magnetic resonance imaging (MRI) at age 6 years showed no obvious brain anomalies. Genetic redundancy between the three members of the B1 subfamily of SOX proteins during early human brain development likely explains the apparently normal development of brain structures in our patient who is nullisomic for SOX3.

Clinical and Biochemical Characteristics of Untreated Adult Patients With Resistance to Thyroid Hormone Alpha.

Background: Thyroid hormone resistance due to pathogenic variants in thyroid hormone receptor alpha (THRA) is rare and descriptions of patients are sparse. The disorder is probably underdiagnosed as patients may have normal thyroid function tests. Treatment with thyroxine in childhood improves clinical symptoms. However, it is not clear if treatment has beneficial effects if started in adulthood. Cases: We investigated 4 previously untreated Caucasian adult first-degree-related patients with the THRA c.788C > T, p.(Ala263Val) variant identified by a gene panel for intellectual disability in the index patient. Clinical data and previous investigations were obtained from medical reports. Results: During childhood and adolescence, short stature, short limbs, metacarpals, and phalanges, and delayed bone age maturation were observed. Delayed motor and language development and decreased intellectual and learning abilities were described. Abdominal adiposity, round face, and increased head circumference were common features. All individuals complained of tiredness, constipation, and low mood. While thyrotropin (TSH) and free thyroxine (FT4) were within the reference range, free triiodothyronine (FT3) was high. FT4/FT3 ratio and reverse T3 were low. Other main features were low hemoglobin and high LDL/HDL ratio. Conclusion: Investigation of 4 first-degree-related adult patients with untreated resistance to thyroid hormone alpha (RTHα) revealed more pronounced phenotype features and hypothyroid symptoms than previously described in patients treated with levothyroxine from childhood or adolescence. The delay in diagnosis is probably due to normal thyroid function tests. We suggest that THRA analysis should be performed in patients with specific clinical features, as treatment in early childhood may improve outcomes.

A novel homozygous variant in the SPG7 gene presenting with childhood optic nerve atrophy.

PURPOSE: To describe a case of hereditary spastic ataxia (HSP) presenting with childhood optic nerve atrophy and report a novel homozygous variant in the SPG7 gene. OBSERVATIONS: A 57-year-old man suffering from progressive optic nerve atrophy since childhood eventually underwent genetic testing. A targeted whole exome gene sequencing panel for optic neuropathy identified a novel homozygous variant in the SPG7 gene, c.2T > G, p.(Met?), which likely abolished production of paraplegin, an inner mitochondrial membrane protein. Subsequent neurologic examination revealed subtle signs of spastic paraplegia and ataxia in keeping with the genetic diagnosis of SPG7. CONCLUSION AND IMPORTANCE: Spastic paraplegia 7 (SPG7) is an autosomal recessive form of the neurodegenerative disorder HSP. Pure HSP is characterized by spastic paraparesis in the lower limbs, whereas complicated HSP presents additional neurological manifestations. This case report adds to the evidence that SPG7 can present with childhood optic nerve atrophy, preceding the characteristic SPG7 manifestations. SPG7 should be considered in the workup of suspected hereditary optic neuropathy.

[Structural variation in the human genome contributes to variation of traits]. / Strukturell variasjon i genomet bidrar til variasjon i egenskaper.

BACKGROUND: The human genome contains a large number of single nucleotide polymorphisms (SNPs) that contribute to normal variation of human traits. Many SNPs have also been shown to affect the development and predisposition of disease. Comparisons of genomes have recently shown that also large DNA segments can vary in structure and number of copies between individuals. A large number of duplications, deletions and inversions has been detected, ranging in size from a few thousand to several millions base pairs. Here we describe the structural variations detected in the human genome, their impact on normal phenotypic variation in the population, and how differences in genome structure may contribute to development of disease. MATERIAL AND METHODS: This article is based on studies of literature retrieved through a non-systematic search of PubMed. RESULTS AND INTERPRETATION: Many structural variations in the genome overlap with genes. Duplications and deletions may change the copy number of genes, and inversions may disrupt gene structure. Some of the affected genes contribute to phenotypic variation between healthy individuals, while others can predispose to disease or contribute to disease development. Diseases caused by such structural variations constitute a major health problem in the population.

Human TGF-ß1 deficiency causes severe inflammatory bowel disease and encephalopathy.

Transforming growth factor (TGF)-ß1 (encoded by TGFB1) is the prototypic member of the TGF-ß family of 33 proteins that orchestrate embryogenesis, development and tissue homeostasis1,2. Following its discovery 3 , enormous interest and numerous controversies have emerged about the role of TGF-ß in coordinating the balance of pro- and anti-oncogenic properties4,5, pro- and anti-inflammatory effects 6 , or pro- and anti-fibrinogenic characteristics 7 . Here we describe three individuals from two pedigrees with biallelic loss-of-function mutations in the TGFB1 gene who presented with severe infantile inflammatory bowel disease (IBD) and central nervous system (CNS) disease associated with epilepsy, brain atrophy and posterior leukoencephalopathy. The proteins encoded by the mutated TGFB1 alleles were characterized by impaired secretion, function or stability of the TGF-ß1-LAP complex, which is suggestive of perturbed bioavailability of TGF-ß1. Our study shows that TGF-ß1 has a critical and nonredundant role in the development and homeostasis of intestinal immunity and the CNS in humans.

Novel PIGT Variant in Two Brothers: Expansion of the Multiple Congenital Anomalies-Hypotonia Seizures Syndrome 3 Phenotype.

Biallelic PIGT variants were previously reported in seven patients from three families with Multiple Congenital Anomalies-Hypotonia Seizures Syndrome 3 (MCAHS3), characterized by epileptic encephalopathy, hypotonia, global developmental delay/intellectual disability, cerebral and cerebellar atrophy, craniofacial dysmorphisms, and skeletal, ophthalmological, cardiac, and genitourinary abnormalities. We report a novel homozygous PIGT missense variant c.1079G>T (p.Gly360Val) in two brothers with several of the typical features of MCAHS3, but in addition, pyramidal tract neurological signs. Notably, they are the first patients with MCAHS3 without skeletal, cardiac, or genitourinary anomalies. PIGT encodes a crucial subunit of the glycosylphosphatidylinositol (GPI) transamidase complex, which catalyzes the attachment of proteins to GPI-anchors, attaching the proteins to the cell membrane. In vitro studies in cells from the two brothers showed reduced levels of GPI-anchors and GPI-anchored proteins on the cell surface, supporting the pathogenicity of the novel PIGT variant.

Clinical and molecular characteristics in three families with biallelic mutations in IGHMBP2.

Biallelic mutations in IGHMBP2 cause spinal muscular atrophy with respiratory distress type 1 (SMARD1) or Charcot-Marie-Tooth type 2S (CMT2S). We report three families variably affected by IGHMBP2 mutations. Patient 1, an 8-year-old boy with two homozygous variants: c.2T>C and c.861C>G, was wheelchair bound due to sensorimotor axonal neuropathy and chronic respiratory failure. Patient 2 and his younger sister, Patient 3, had compound heterozygous variants: c.983_987delAAGAA and c.1478C>T. However, clinical phenotypes differed markedly as the elder with sensorimotor axonal neuropathy had still unaffected respiratory function at 4.5 years, whereas the younger presented as infantile spinal muscular atrophy and died from relentless respiratory failure at 11 months. Patient 4, a 6-year-old girl homozygous for IGHMBP2 c.449+1G>T documented to result in two aberrant transcripts, was wheelchair dependent due to axonal polyneuropathy. The clinical presentation in Patients 1 and 3 were consistent with SMARD1, whereas Patients 2 and 4 were in agreement with CMT2S.

A novel mutation in FBXL4 in a Norwegian child with encephalomyopathic mitochondrial DNA depletion syndrome 13.

Mitochondrial DNA depletion syndromes (MTDPS) represent a clinically and genetically heterogeneous group of autosomal recessive disorders, caused by mutations in genes involved in maintenance of mitochondrial DNA (mtDNA). Biallelic mutations in FBXL4 were recently described to cause encephalomyopathic MTDPS13. The syndrome has infantile onset and presents with hypotonia, feeding difficulties, a pattern of mild facial dysmorphisms, global developmental delay and brain atrophy. Laboratory investigations reveal elevated blood lactate levels, unspecific mitochondrial respiratory chain (MRC) enzyme deficiencies and mtDNA depletion. We report a novel missense variant, c.1442T > C (p.Leu481Pro), in FBXL4 (NM_012160.4) in a Norwegian boy with clinical, biochemical and cerebral MRI characteristics consistent with MTDPS13. The FBXL4 c.1442T > C (p.Leu481Pro) variant was not present in public databases, 149 Norwegian controls nor an in-house database containing whole exome sequencing data from 440 individuals, and it was predicted in silico to be deleterious to the protein function. Activities of MRC enzymes were normal in muscle tissue (complexes I-IV) and cultured skin fibroblasts (complexes I-V) from the patient, but mtDNA depletion was confirmed in muscle, thus supporting the predicted pathogenicity of the FBXL4 c.1442T > C (p.Leu481Pro) variant. On clinical indication of mitochondrial encephalomyopathy, sequencing of FBXL4 should be performed, even when the activity levels of the MRC enzymes are normal.

A recurrent deletion on chromosome 2q13 is associated with developmental delay and mild facial dysmorphisms.

We report two unrelated patients with overlapping chromosome 2q13 deletions (patient 1 in chr2:111415137-113194067 bp and patient 2 in chr2:110980342-113007823 bp, hg 19). Patient 1 presents with developmental delay, microcephaly and mild dysmorphic facial features, and patient 2 with autism spectrum disorder, borderline cognitive abilities, deficits in attention and executive functions and mild dysmorphic facial features. The mother and maternal grandmother of patient 1 were healthy carriers of the deletion. Previously, 2q13 deletions were reported in 27 patients, and the interpretation of its clinical significance varied. Our findings support that the 2q13 deletion is associated with a developmental delay syndrome manifesting with variable expressivity and reduced penetrance which poses a challenge for genetic counselling as well as the clinical recognition of 2q13 deletion patients.

Haploinsufficiency of XPO1 and USP34 by a de novo 230 kb deletion in 2p15, in a patient with mild intellectual disability and cranio-facial dysmorphisms.

2p15p16.1-deletion syndrome was first described in 2007 based on the clinical presentation of two patients. The syndrome is characterized by intellectual disability, autism spectrum disorders, microcephaly, dysmorphic facial features and a variety of congenital organ defects. The precise genotype-phenotype correlation in 2p15-deletion syndrome is not understood. However, greater insight can be obtained by thorough clinical investigation of patients carrying deletions, especially those of small size. We report a 21-year-old male patient with features overlapping the clinical spectrum of the 2p15p16.1-deletion syndrome, such as intellectual disability, dysmorphic facial features, and congenital defects. He carried a 230 kb de novo deletion (chr2:61500346-61733075 bp, hg19), which affects the genes USP34, SNORA70B and XPO1. While there is a lack of functional data on SNORA70B, the involvement of USP34 and XPO1 in the regulation of fundamental developmental processes is well known. We suggest that haploinsufficiency of one or both of these genes is likely to be responsible for the disease in our patient.

Haploinsufficiency of two histone modifier genes on 6p22.3, ATXN1 and JARID2, is associated with intellectual disability.

BACKGROUND: Nineteen patients with deletions in chromosome 6p22-p24 have been published so far. The syndromic phenotype is varied, and includes intellectual disability, behavioural abnormalities, dysmorphic features and structural organ defects. Heterogeneous deletion breakpoints and sizes (1-17 Mb) and overlapping phenotypes have made the identification of the disease causing genes challenging. We suggest JARID2 and ATXN1, both harbored in 6p22.3, as disease causing genes. METHODS AND RESULTS: We describe five unrelated patients with de novo deletions (0.1-4.8 Mb in size) in chromosome 6p22.3-p24.1 detected by aCGH in a cohort of approximately 3600 patients ascertained for neurodevelopmental disorders. Two patients (Patients 4 and 5) carried non-overlapping deletions that were encompassed by the deletions of the remaining three patients (Patients 1-3), indicating the existence of two distinct dosage sensitive genes responsible for impaired cognitive function in 6p22.3 deletion-patients. The smallest region of overlap (SRO I) in Patients 1-4 (189 kb) included the genes JARID2 and DTNBP1, while SRO II in Patients 1-3 and 5 (116 kb) contained GMPR and ATXN1. Patients with deletion of SRO I manifested variable degrees of cognitive impairment, gait disturbance and distinct, similar facial dysmorphic features (prominent supraorbital ridges, deep set eyes, dark infraorbital circles and midface hypoplasia) that might be ascribed to the haploinsufficiency of JARID2. Patients with deletion of SRO II showed intellectual disability and behavioural abnormalities, likely to be caused by the deletion of ATXN1. Patients 1-3 presented with lower cognitive function than Patients 4 and 5, possibly due to the concomitant haploinsufficiency of both ATXN1 and JARID2. The chromatin modifier genes ATXN1 and JARID2 are likely candidates contributing to the clinical phenotype in 6p22-p24 deletion-patients. Both genes exert their effect on the Notch signalling pathway, which plays an important role in several developmental processes. CONCLUSIONS: Patients carrying JARID2 deletion manifested with cognitive impairment, gait disturbance and a characteristic facial appearance, whereas patients with deletion of ATXN1 seemed to be characterized by intellectual disability and behavioural abnormalities. Due to the characteristic facial appearance, JARID2 haploinsufficiency might represent a clinically recognizable neurodevelopmental syndrome.

A 1 Mb de novo deletion within 11q13.1q13.2 in a boy with mild intellectual disability and minor dysmorphic features.

We report a 11 year old male patient ascertained for mild intellectual disability and minor dysmorphic features, carrying a 1 Mb de novo deletion on chromosome 11q13.1q13.2 detected by aCGH. This is the first report of a deletion in this region in a patient presenting with intellectual impairment and mild dysmorphic traits. The 1 Mb deleted area encompasses 47 RefSeq genes, including Cornichon homologue 2 (CNIH2), Cofilin-1 (CFL1) and neuronal PAS domain-containing protein 4 (NPAS4), which are highly expressed in the central nervous system. Knockout of the CNIH2 and CFL1 orthologues in animals results in migration disturbances, while low or no expression of Npas4 in mice results in impairment of memory and learning. These three genes have previously been suggested as candidate genes for neurological disorders.

shRNA expression constructs designed directly from siRNA oligonucleotide sequences.

Gene silencing by RNA interference (RNAi) is a widely used approach for target-specific knockdown of gene expression. Induction of RNAi in mammalian cells can be achieved by introduction of synthetic small interfering RNA (siRNA) resulting in transient knockdown, or alternatively by stable expression of short hairpin RNA (shRNA). Several algorithms for efficient siRNA design exist, but recent reports have suggested that these cannot be directly used to design efficient shRNAs. In this study, 25 siRNAs targeting independent sequences in five transcripts were used for the construction of shRNA cassettes. Both the siRNAs and shRNA constructs were transfected into HEK293T cells. Quantitative real-time PCR analysis revealed that 19 out of the 25 shRNA constructs reduced the average expression level to less than 30%. Our data support that sequences designed by siRNA algorithms efficiently reduce the expression of the target gene when converted into shRNA expression constructs.

A de novo 15q13.2q13.3 deletion in a boy with an Angelman syndrome like phenotype.

We report on a 11-year-old boy investigated for a clinical suspicion of Angelman syndrome (AS) (OMIM 105830) who was found to carry a de novo interstitial deletion of chromosome 15q13.2q13.3. The deletion overlaps the critical region for the newly recognized recurrent 15q13.3 deletion syndrome. This is the first report of a patient with 15q13.3 deletion syndrome with clinical features similar to that of AS, thus broadening the phenotypic spectrum associated with the 15q13.3 microdeletion syndrome.