Phenotypic and mutational spectrum of ROR2-related Robinow syndrome.

Robinow syndrome is characterized by a triad of craniofacial dysmorphisms, disproportionate-limb short stature, and genital hypoplasia. A significant degree of phenotypic variability seems to correlate with different genes/loci. Disturbances of the noncanonical WNT-pathway have been identified as the main cause of the syndrome. Biallelic variants in ROR2 cause an autosomal recessive form of the syndrome with distinctive skeletal findings. Twenty-two patients with a clinical diagnosis of autosomal recessive Robinow syndrome were screened for variants in ROR2 using multiple molecular approaches. We identified 25 putatively pathogenic ROR2 variants, 16 novel, including single nucleotide variants and exonic deletions. Detailed phenotypic analyses revealed that all subjects presented with a prominent forehead, hypertelorism, short nose, abnormality of the nasal tip, brachydactyly, mesomelic limb shortening, short stature, and genital hypoplasia in male patients. A total of 19 clinical features were present in more than 75% of the subjects, thus pointing to an overall uniformity of the phenotype. Disease-causing variants in ROR2, contribute to a clinically recognizable autosomal recessive trait phenotype with multiple skeletal defects. A comprehensive quantitative clinical evaluation of this cohort delineated the phenotypic spectrum of ROR2-related Robinow syndrome. The identification of exonic deletion variant alleles further supports the contention of a loss-of-function mechanism in the etiology of the syndrome.

Bi-allelic TTC5 variants cause delayed developmental milestones and intellectual disability.

BACKGROUND: Intellectual disability syndromes (IDSs) with or without developmental delays affect up to 3% of the world population. We sought to clinically and genetically characterise a novel IDS segregating in five unrelated consanguineous families. METHODS: Clinical analyses were performed for eight patients with intellectual disability (ID). Whole-exome sequencing for selected participants followed by Sanger sequencing for all available family members was completed. Identity-by-descent (IBD) mapping was carried out for patients in two Egyptian families harbouring an identical variant. RNA was extracted from blood cells of Turkish participants, followed by cDNA synthesis and real-time PCR for TTC5. RESULTS: Phenotype comparisons of patients revealed shared clinical features of moderate-to-severe ID, corpus callosum agenesis, mild ventriculomegaly, simplified gyral pattern, cerebral atrophy, delayed motor and verbal milestones and hypotonia, presenting with an IDS. Four novel homozygous variants in TTC5: c.629A>G;p.(Tyr210Cys), c.692C>T;p.(Ala231Val), c.787C>T;p.(Arg263Ter) and c.1883C>T;p.(Arg395Ter) were identified in the eight patients from participating families. IBD mapping revealed that c.787C>T;p.(Arg263Ter) is a founder variant in Egypt. Missense variants c.629A>G;p.(Tyr210Cys) and c.692C>T;p.(Ala231Val) disrupt highly conserved residues of TTC5 within the fifth and sixth tetratricopeptide repeat motifs which are required for p300 interaction, while the nonsense variants are predicted to decrease TTC5 expression. Functional analysis of variant c.1883C>T;p.(Arg395Ter) showed reduced TTC5 transcript levels in accordance with nonsense-mediated decay. CONCLUSION: Combining our clinical and molecular data with a recent case report, we identify the core and variable clinical features associated with TTC5 loss-of-function variants and reveal the requirement for TTC5 in human brain development and health.

Clinical and exome sequencing findings in seven children with Bardet-Biedl syndrome from Turkey.

BACKGROUND: Bardet-Biedl syndrome (BBS) is a very-rare autosomal recessive genetic disorder with severe multisystem manifestations. Genetic testing plays an important role in the early diagnosis of the disease. In this study, while trying to elucidate the genetic etiology of seven individuals with clinical BBS diagnosis from six different families, we also aimed to examine the distribution of BBS variations in this region of Turkey. METHODS AND MATERIALS: Exome sequencing analysis is performed for clinically diagnosed patients with BBS in the present study followed by parental segregation. The unreported and previously described clinical features are presented. RESULTS: Homozygous variants, four of which are unreported, in BBS-related genes (BBS5 [c.682-2A > G], MKKS [c.775del], BBS7 [c.849+1G > T], BBS9 [c.965G > A], BBS10 [c.145C > T], LZTFL1[c.384G > A]) are detected for all the seven individuals included in the study. The most common clinical finding is polydactyly followed by renal anomalies. The clinical features not previously described are correlated to the unreported variant. CONCLUSIONS: In this study, exome sequencing findings are discussed and four previously unreported disease-associated variants are described including the fifth BBS-implicated LZTFL1 change and possible genotype-phenotype correlation is described.

An epilepsy-associated ACTL6B variant captures neuronal hyperexcitability in a human induced pluripotent stem cell model.

ACTL6B is a component of the neuronal BRG1/brm-associated factor (nBAF) complex, which is required for chromatin remodeling in postmitotic neurons. We recently reported biallelic pathogenic variants in ACTL6B in patients diagnosed with early infantile epileptic encephalopathy, subtype 76 (EIEE-76), presenting with severe, global developmental delay, epileptic encephalopathy, cerebral atrophy, and abnormal central nervous system myelination. However, the pathophysiological mechanisms underlying their phenotype is unknown. Here, we investigate the molecular pathogenesis of ACTL6B p.(Val421_Cys425del) using in silico 3D protein modeling predictions and patient-specific induced pluripotent stem cell-derived neurons. We found neurons derived from EIEE-76 patients showed impaired accumulation of ACTL6B compared to unaffected relatives, caused by reduced protein stability. Furthermore, EIEE-76 patient-derived neurons had dysregulated nBAF target gene expression, including genes important for neuronal development and disease. Multielectrode array system analysis unveiled elevated electrophysiological activity of EIEE-76 patients-derived neurons, consistent with the patient phenotype. Taken together, our findings validate a new model for EIEE-76 and reveal how reduced ACTL6B expression affects neuronal function.

A novel homozygous RIPK4 variant in a family with severe Bartsocas-Papas syndrome.

Bartsocas-Papas syndrome (BPS) is a rare autosomal recessive disorder characterized by popliteal pterygia, syndactyly, ankyloblepharon, filiform bands between the jaws, cleft lip and palate, and genital malformations. Most of the BPS cases reported to date are fatal either in the prenatal or neonatal period. Causative genetic defects of BPS were mapped on the RIPK4 gene encoding receptor-interacting serine/threonine kinase 4, which is critical for epidermal differentiation and development. RIPK4 variants are associated with a wide range of clinical features ranging from milder ectodermal dysplasia to severe BPS. Here, we evaluated a consanguineous Turkish family, who had two pregnancies with severe multiple malformations compatible with BPS phenotype. In order to identify the underlying genetic defect, direct sequencing of the coding region and exon-intron boundaries of RIPK4 was carried out. A homozygous transversion (c.481G>C) that leads to the substitution of a conserved aspartic acid to histidine (p.Asp161His) in the kinase domain of the protein was detected. Pathogenicity predictions, molecular modeling, and cell-based functional assays showed that Asp161 residue is required for the kinase activity of the protein, which indicates that the identified variant is responsible for the severe BPS phenotype in the family.

Expanding the genotypic and phenotypic spectrum of severe serine biosynthesis disorders.

Serine biosynthesis disorders comprise a spectrum of very rare autosomal recessive inborn errors of metabolism with wide phenotypic variability. Neu-Laxova syndrome represents the most severe expression and is characterized by multiple congenital anomalies and pre- or perinatal lethality. Here, we present the mutation spectrum and a detailed phenotypic analysis in 15 unrelated families with severe types of serine biosynthesis disorders. We identified likely disease-causing variants in the PHGDH and PSAT1 genes, several of which have not been reported previously. Phenotype analysis and a comprehensive review of the literature corroborates the evidence that serine biosynthesis disorders represent a continuum with varying degrees of phenotypic expression and suggest that even gradual differences at the severe end of the spectrum may be correlated with particular genotypes. We postulate that the individual residual enzyme activity of mutant proteins is the major determinant of the phenotypic variability, but further functional studies are needed to explore effects at the enzyme protein level.

Responsiveness of sphingosine phosphate lyase insufficiency syndrome to vitamin B6 cofactor supplementation.

Sphingosine-1-phosphate (S1P) lyase is a vitamin B6-dependent enzyme that degrades sphingosine-1-phosphate in the final step of sphingolipid metabolism. In 2017, a new inherited disorder was described caused by mutations in SGPL1, which encodes sphingosine phosphate lyase (SPL). This condition is referred to as SPL insufficiency syndrome (SPLIS) or alternatively as nephrotic syndrome type 14 (NPHS14). Patients with SPLIS exhibit lymphopenia, nephrosis, adrenal insufficiency, and/or neurological defects. No targeted therapy for SPLIS has been reported. Vitamin B6 supplementation has therapeutic activity in some genetic diseases involving B6-dependent enzymes, a finding ascribed largely to the vitamin's chaperone function. We investigated whether B6 supplementation might have activity in SPLIS patients. We retrospectively monitored responses of disease biomarkers in patients supplemented with B6 and measured SPL activity and sphingolipids in B6-treated patient-derived fibroblasts. In two patients, disease biomarkers responded to B6 supplementation. S1P abundance and activity levels increased and sphingolipids decreased in response to B6. One responsive patient is homozygous for an SPL R222Q variant present in almost 30% of SPLIS patients. Molecular modeling suggests the variant distorts the dimer interface which could be overcome by cofactor supplementation. We demonstrate the first potential targeted therapy for SPLIS and suggest that 30% of SPLIS patients might respond to cofactor supplementation.

A Novel AIPL1 Nonsense Mutation: Case Report of Three Siblings Diagnosed with Leber Congenital Amaurosis.

Background: Leber congenital amaurosis (LCA) is a subgroup of early onset retinal dystrophy, manifesting with early or congenital visual loss, wandering nystagmus, amaurotic pupils, oculodigital sign, reduced retinal thickness on optical coherence tomography and abnormal electroretinogram. Today, mutations of about 25 genes account for 80% of individuals with LCA. The AIPL1 mutations causing LCA type 4 account for about 5-10% of this group. Case Report: Three affected siblings with vision loss, nystagmus, cataracts, stage 4 keratoconus, retinal abnormalities (black spots), lack of glaucoma, and dysmorphic features from a consanguineous marriage had LCA type 4 with a novel homozygous missense mutations of AIPL1(c.862 C > T). Conclusion: Cortical cataracts, stage 4 keratoconus, retinal black spots, and lack of glaucoma along with mutations of AIPL1 (c.862 C > T) can be present in LCA type 4.

Pathogenic homozygous variations in ACTL6B cause DECAM syndrome: Developmental delay, Epileptic encephalopathy, Cerebral Atrophy, and abnormal Myelination.

The extensive usage of next generation sequencing, particularly for the patients affected with neurodevelopmental disorders, has increased our understanding and enabled identifying novel disorder genes. Here, we report an extended consanguineous family having at least three affected children with ACTL6B-related neurodevelopmental disorder and expand the known phenotypic spectrum by characterizing the clinical findings using a standardized vocabulary, Human Phenotype Ontology Terms.

A Hemizygous 370 Kilobase Microduplication at Xq13.1 in a Three-Year-Old Boy With Autism and Speech Delay.

BACKGROUND: Alterations of Neuroligin 3 (NLGN3), located on Xq13, have been reported in autism spectrum disorder (ASD), and include the less frequent Xq13 duplication. CASE REPORT: A boy with an aggressive behavior, no speech and weak social relationships had a de novo Xq13.1 microduplication detected by microarray analysis. CONCLUSION: NLGN3, TAF1, and MED12 alterations, located on Xq13.1, have been associated with ASD. TAF and MED12 have other clinical features not present in our case. This supports that duplication of NLGN3 may be associated with ASD.

The powerful association of angiotensin-converting enzyme insertion/deletion polymorphism and idiopathic recurrent pregnancy loss.

OBJECTIVES: Idiopathic recurrent pregnancy loss (IRPL) is one of the most troublesome complications of pregnancy. Several researches were also conducted to search the possible association with ACE I/D polymorphism and IRPL. In the light of these reports, this case-control study was investigated to genotypes and alleles of ACE I/D polymorphism in IRPL group and control group. MATERIAL AND METHODS: Overall, 1176 subjects (1007 cases, 169 controls) were investigated. Allele genotype distributions were determined by PCR method in both groups. Differences in genotype and allele frequencies between groups were investigated by Pearson chi-square tests. The odds ratio (OR) and 95% confidence intervals (95% CI) were also determined. RESULTS: For the ACE I/D polymorphism I and D allele frequencies were in the control and case groups respectively; 49.4 and 41.6%, 50.6 and 58.4%. The genotypes of ACE for I/D observed in control and case group respectively were as follows; II (27.2 and 17.9), ID (44.4 and 47.4) and DD (28.4 and 34.7). Regarding the distribution of D allele and genotypes containing D allele, we observed significant statistical differences between case and control groups. CONCLUSIONS: Our results showed that the ACE I/D polymorphism was associated with IRPL, and that women that carried DD or ID genotypes had a 72% elevated risk of developing IRPL than women with the II genotype (OR (95% CI): 1.72 (1.181-2.5)). This odds ratio was found to be 1.61 in a case-control study and 1.28 in a meta-analysis study compiling 11 separate studies, which is consistent with our study data.

A Genotyped Case of Townes-Brocks Syndrome with Absent Pulmonary Valve Syndrome from Turkey.

Townes-Brocks syndrome (TBS) is a rare syndrome characterized by triad of anal, ear, and thumb anomalies. Further malformations/anomalies include congenital heart diseases, foot malformations, sensorineural and/or conductive hearing impairment, genitourinary malformations, and anomalies of eye and nervous system. Definitive diagnosis for TBS is confirmed by molecular analysis for mutations in the SALL1 gene. Only one known case of TBS with absent pulmonary valve syndrome (APVS) has been previously described to our knowledge. Here, we report a newborn diagnosed with TBS with APVS and tetralogy of Fallot (TOF) who was found to carry the most common pathogenic SALL1 gene mutation c.826C > T (p.R276X), with its surgical repair and postoperative follow-up. To our knowledge, this is the first genotyped case of TBS from Turkey to date. TBS should be suspected in the presence of ear, anal, and thumb malformations in a neonate. If a patient with TBS and TOF-APVS needs preoperative ventilation within the first months of life, this implies prolonged postoperative intubation and increased risk of mortality.

A novel COQ7 mutation causing primarily neuromuscular pathology and its treatment options.

Coenzyme Q10 (CoQ10) is necessary as electron transporter in mitochondrial respiration and other cellular functions. CoQ10 is synthesized by all cells and defects in the synthesis pathway result in primary CoQ10 deficiency that frequently leads to severe mitochondrial disease syndrome. CoQ10 is exceedingly hydrophobic, insoluble, and poorly bioavailable, with the result that dietary CoQ10 supplementation produces no or only minimal relief for patients. We studied a patient from Turkey and identified and characterized a new mutation in the CoQ10 biosynthetic gene COQ7 (c.161G > A; p.Arg54Gln). We find that unexpected neuromuscular pathology can accompany CoQ10 deficiency caused by a COQ7 mutation. We also show that by-passing the need for COQ7 by providing the unnatural precursor 2,4-dihydroxybenzoic acid, as has been proposed, is unlikely to be an effective and safe therapeutic option. In contrast, we show for the first time in human patient cells that the respiratory defect resulting from CoQ10 deficiency is rescued by providing CoQ10 formulated with caspofungin (CF/CoQ). Caspofungin is a clinically approved intravenous fungicide whose surfactant properties lead to CoQ10 micellization, complete water solubilization, and efficient uptake by cells and organs in animal studies. These findings reinforce the possibility of using CF/CoQ in the clinical treatment of CoQ10-deficient patients.

A Newborn with Infantile-Onset Pompe Disease Improving after Administration of Enzyme Replacement Therapy: Case Report.

Pompe disease (PD) is an autosomal recessive lysosomal storage disorder caused by a deficiency of acid α-1,4-glucosidase enzyme (GAA). PD has two forms, namely the infantile-onset and the late-onset form. In untreated cases, infantile-onset form usually leads to cardio-respiratory failure and death in the first year of life. Herein, we report a newborn with infantile-onset PD characterized by muscular hypotonia, respiratory distress, hypertrophic cardiomyopathy, hepatomegaly, elevated serum enzyme levels of aspartate aminotransferase of 117 IU/L (three times the normal value), alanine aminotransferase of 66 IU/L (1.8 times the normal value), lactate dehydrogenase of 558 IU/L (1.2 times the normal value), and creatine kinase >5,000 IU/L (16 times the normal value). Dried blood spot testing was performed and revealed decreased GAA enzymatic activity (0.07 nmol/mL/h, normal 0.93-7.33 nmol/mL/h). GAA gene analysis performed for confirming the diagnosis showed homozygous mutation c.896T >C (p.Leu299Pro). Initiation of enzyme replacement therapy (ERT) (ERT; 20 mg/kg, once every week) at 28 days of age resulted in weaning off from respiratory support within 1 week after treatment, normalization of cardiac abnormalities, and normal neuromotor development in the 16th month of age. Early diagnosis and early treatment with ERT, especially in the neonatal period, is of great importance to improve cardiac function and motor development in infantile-onset PD.

Congenital insensitivity to pain: a novel mutation affecting a U12-type intron causes multiple aberrant splicing of SCN9A.

ABSTRACT: Mutations in the alpha subunit of voltage-gated sodium channel 1.7 (NaV1.7), encoded by SCN9A gene, play an important role in the regulation of nociception and can lead to a wide range of clinical outcomes, ranging from extreme pain syndromes to congenital inability to experience pain. To expand the phenotypic and genotypic spectrum of SCN9A-related channelopathies, we describe the proband, a daughter born from consanguineous parents, who had pain insensitivity, diminished temperature sensation, foot burns, and severe loss of nociceptive nerve fibers in the epidermis. Next-generation sequencing of SCN9A (NM_002977.3) revealed a novel homozygous substitution (c.377+7T>G) in the donor splice site of intron 3. As the RNA functional testing is challenging, the in silico analysis is the first approach to predict possible alterations. In this case, the computational analysis was unable to identify the splicing consensus and could not provide any prediction for splicing defects. The affected intron indeed belongs to the U12 type, a family of introns characterised by noncanonical consensus at the splice sites, accounting only for 0.35% of all human introns, and is not included in most of the training sets for splicing prediction. A functional study on proband RNA showed different aberrant transcripts, where exon 3 was missing and an intron fragment was included. A quantification study using real-time polymerase chain reaction showed a significant reduction of the NaV1.7 canonical transcript. Collectively, these data widen the spectrum of SCN9A-related insensitivity to pain by describing a mutation causing NaV1.7 deficiency, underlying the nociceptor dysfunction, and highlight the importance of molecular investigation of U12 introns' mutations despite the silent prediction.

Suppression of premature transcription termination leads to reduced mRNA isoform diversity and neurodegeneration.

Tight regulation of mRNA isoform expression is essential for neuronal development, maintenance, and function; however, the repertoire of proteins that govern isoform composition and abundance remains incomplete. Here, we show that the RNA kinase CLP1 regulates mRNA isoform expression through suppression of proximal cleavage and polyadenylation. We found that human stem-cell-derived motor neurons without CLP1 or with the disease-associated CLP1 p.R140H variant had distinct patterns of RNA-polymerase-II-associated cleavage and polyadenylation complex proteins that correlated with polyadenylation site usage. These changes resulted in imbalanced mRNA isoform expression of long genes important for neuronal function that were recapitulated in vivo. Strikingly, we observed the same pattern of reduced mRNA isoform diversity in 3' end sequencing data from brain tissues of patients with neurodegenerative disease. Together, our results identify a previously uncharacterized role for CLP1 in mRNA 3' end formation and reveal an mRNA misprocessing signature in neurodegeneration that may suggest a common mechanism of disease.

Clinical findings and a DNA methylation signature in kindreds with alterations in ZNF711.

ZNF711 is one of eleven zinc-finger genes on the X chromosome that have been associated with X-linked intellectual disability. This association is confirmed by the clinical findings in 20 new cases in addition to 11 cases previously reported. No consistent growth aberrations, craniofacial dysmorphology, malformations or neurologic findings are associated with alterations in ZNF711. The intellectual disability is typically mild and coexisting autism occurs in half of the cases. Carrier females show no manifestations. A ZNF711-specific methylation signature has been identified which can assist in identifying new cases and in confirming the pathogenicity of variants in the gene.

A different look at genetic factors in individuals with non-obstructive azoospermia or oligospermia in our research study: To whom, which threshold, when, in what way?

INTRODUCTION: In our study, we sought answers to many questions about male infertility from a different perspective. The first step in male infertility is anamnesis, physical examination and sperm count. The European Academy of Andrology recommends examination of genetic causes in individuals with fewer than 5million/ml semen counts. The American Urological Association and American Society for Reproductive Medicine have guidelines recommending performing karyotype and AZF subgroup deletion testing in azoospermia and fewer than 5 million sperm total count. Klinefelter syndrome and Y chromosome microdeletions are still very important in male infertility. Based on patients with Klinefelter syndrome or Y microdeletion, we sought answers to many questions in male infertility. MATERIALS AND METHODS: In the presented study 327 male patients with having fewer than 15millionsperm/ml detected in at least two consecutive sperm analysis were examined. Patients were divided into sub-groups according to the presence of semen count, chromosomal anomaly and Y microdeletion. In addition, FSH, LH and testosterone levels were analyzed. RESULTS: Numerical chromosomal anomalies were observed in 34 (10.4%) of 327 patients, and all of these anomalies were found as 47, XXY. Individuals with no AZF microdeletion constituted 95.1% (n=311) of the study group. The overall frequency of AZF microdeletions was 4.9% (16/327). No AZF microdeletions were detected for the patients who have sperm counts above 2million/ml. FSH, LH and testosterone levels were found significantly different between the groups. DISCUSSION: The results of our study provide another layer of evidence to demonstrate the controversial threshold value of the EAA. In light of our data and current literature, we recommend to set the threshold value at 2million/ml for semen analysis. Further studies conducted in different ethnic groups and larger patient groups would contribute to clarify what exact value should be used to apply genetic tests.

The Association Between BMP-2, UQCC1 and CX3CR1 Polymorphisms and the Risk of Developmental Dysplasia of the Hip.

OBJECTIVE: Developmental dysplasia of the hip (DDH) is a complicated skeletal disease ranging from subluxation to complete dislocation of the hip as a result of insufficient development of the acetabulum and femur. To date, numerous genes such as C-X3-C motif chemokine receptor 1 (CX3CR1), ubiquinol-cytochrome c reductase complex assembly factor 1 (UQCC1) and growth/differentiation factor 5 (GDF5), have been investigated to elucidate the underlying genetic etiology. Turkish population is one of the communities where DDH patients frequently observed, but almost no study has been conducted to elucidate the genetic etiology. In our study, we aimed to investigate the polymorphism of CX3CR1 rs3732378 and UQCC1 rs6060373, which have been shown to be associated with DDH in different populations. In addition, we aimed to investigate the BMP-2 rs235768 polymorphism which has not been investigated in the etiology of DDH. METHODS: Overall, 168 subjects (68 participants in the patient group, 100 participants in the control group) were investigated. The participants with following evidence and symptoms were excluded from the two groups: any systemic syndrome, another congenital anomaly, hereditary diseases, breech presentation, history of oligohydramnios, swaddling and high birth weight (> 4000 g). 3 single-nucleotide polymorphisms (SNP) were examined by qRT-PCR method. RESULTS: For CX3CR1 rs3732378 polymorphism, significant differences were observed in genotypes and allele frequencies (p < 0.0001). This condition was associated with a 12-fold increased risk in recessive modeling and 75-fold increased risk in dominant modeling. There was no significant relationship between DDH and the other two polymorphisms. CONCLUSIONS: Our work is the first study to investigate DDH and genetic polymorphisms in Turkish population where DDH is observed quite frequently. It is also the first study to investigate the relationship between BMP-2 rs235768 polymorphism and DDH. Our study revealed a clear relationship between CX3CR1 rs3732378 polymorphism and DDH in Turkish population.