Association of the Recurrent Rare Variant c.415T>C p.Phe139Leu in CLN5 With a Recessively Inherited Macular Dystrophy.

Importance: Homozygous variants in the neuronal ceroid lipofuscinosis type 5 (CLN5) gene are associated with neuronal ceroid lipofuscinosis, a progressive neurologic disorder that leads to ataxia, seizures, and early death. The association between a homozygous variant in this gene and a macular dystrophy is described here. Objective: To describe an autosomal recessive macular dystrophy associated with a recurrent variant in CLN5. Design, Setting, and Participants: This cohort study took place at a national referral center and had a follow-up duration ranging between 1 and 5 years. All patients who were identified to carry a specific homozygous missense variant in CLN5, among more than 2000 patients who were diagnosed with or suspected to have retinal dystrophies, who did not carry this variant, were included. Data were collected between June 2014 and September 2020. Exposures: All patients who were sampled for DNA analysis due to molecularly unconfirmed retinal dystrophy and who were subsequently identified to carry the homozygous missense variant c.415T>C (p.Phe139Leu) in CLN5 were included, while patients who did not carry the variant were excluded. Main Outcomes and Measures: Retinal phenotype associated with this specific homozygous missense variant in CLN5. Results: Seven affected patients (mean [SD] age, 43 [18] years; age range, 33-52 years; 5 male) carried the homozygous missense in CLN5. All patients were diagnosed as having a macular dystrophy. Four patients had mild electroretinographic alterations. All patients had hypoautofluorescent maculas with retinal thinning (central subfield thickness, 80 µm). Visual acuity ranged between 2/200 and 20/100. Neurologic symptoms were mild (dizziness) in 5 patients and absent in 2 patients. Neuroimaging demonstrated cerebellar atrophy and white matter lesions, respectively, in 2 patients. Conclusions and Relevance: These results suggest that CLN5, similar to CLN7, may be associated with isolated macular dystrophy as well as neuronal ceroid lipofuscinosis. The variant c.415T>C p.Phe139Leu does not seem to be associated with any prominent neurologic disease at least until the fourth to sixth decades of life. These findings may imply a specific role of CLN5 in macular neurons. Additional study is suggested, such as molecular screening for this variant in cohorts of patients with undiagnosed macular dystrophies and biological studies of its molecular effects.

Severe retinitis pigmentosa phenotype associated with novel CNGB1 variants.

PURPOSE: To report a severe phenotype of retinitis pigmentosa associated with novel mutations in CNGB1. OBSERVATIONS: Six siblings, age range 50-75 years old, were examined using optical coherence tomography and fundus autofluorescene, electroretinogram testing, Goldman visual field testing, and genetic testing using next generation sequencing.In four affected siblings, two novel compound heterozygous variants in CNGB1 were detected: in exon 26 the missense variant c.2603G > A (p.(Gly868Asp)), and in exon 21, the in-frame 12-bp duplication c.2093_2104dupGCGACCTCATCT (p.(Cys698_lle701dup)). One sibling was unaffected and carried neither of the variants, while another sibling had mild macular degeneration changes and carried the latter variant in heterozygous status. The affected siblings presented with a phenotype showing markedly constricted visual field, flat scotopic and photopic electroretinogram responses and generalized retinal atrophy. CONCLUSIONS AND IMPORTANCE: This is the first report of a 12bp in-frame duplication and a missense variant (in compound heterozygous status) in CNGB1, being associated with a severe form of retinitis pigmentosa featuring extensive peripheral and central retinal degeneration. This study expands the molecular genetic basis of CNGB1-related disease.

Next-generation sequencing identifies unexpected genotype-phenotype correlations in patients with retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited degenerative disease causing severe retinal dystrophy and visual impairment mainly with onset in infancy or adolescence. Targeted next-generation sequencing (NGS) has become an efficient tool to encounter the enormous genetic heterogeneity of diverse retinal dystrophies, including RP. To identify disease-causing mutations in unselected, consecutive RP patients, we conducted Sanger sequencing of genes commonly involved in the suspected genetic RP subtype, followed by targeted large-panel NGS if no mutation was identified, or NGS as primary analysis. A high (70%) detection rate of disease-causing mutations was achieved in a large cohort of 116 unrelated patients. About half (48%) of the solved RP cases were explained by mutations in four genes: RPGR, EYS, PRPF31 and USH2A. Overall, 110 different mutations distributed across 30 different genes were detected, and 46 of these mutations were novel. A molecular diagnosis was achieved in the majority (82-100%) of patients if the family history was suggestive for a particular mode of inheritance, but only in 60% in cases of sporadic RP. The diagnostic potential of extensive molecular analysis in a routine setting is also illustrated by the identification of unexpected genotype-phenotype correlations for RP patients with mutations in CRX, CEP290, RPGRIP1, MFSD8. Furthermore, we identified numerous mutations in autosomal dominant (PRPF31, PRPH2, CRX) and X-linked (RPGR) RP genes in patients with sporadic RP. Variants in RP2 and RPGR were also found in female RP patients with apparently sporadic or dominant disease. In summary, this study demonstrates that massively parallel sequencing of all known retinal dystrophy genes is a valuable diagnostic approach for RP patients.

Clinical and genetic characteristics of 251 consecutive patients with macular and cone/cone-rod dystrophy.

Macular and cone/cone-rod dystrophies (MD/CCRD) demonstrate a broad genetic and phenotypic heterogeneity, with retinal alterations solely or predominantly involving the central retina. Targeted next-generation sequencing (NGS) is an efficient diagnostic tool for identifying mutations in patient with retinitis pigmentosa, which shows similar genetic heterogeneity. To detect the genetic causes of disease in patients with MD/CCRD, we implemented a two-tier procedure consisting of Sanger sequencing and targeted NGS including genes associated with clinically overlapping conditions. Disease-causing mutations were identified in 74% of 251 consecutive MD/CCRD patients (33% of the variants were novel). Mutations in ABCA4, PRPH2 and BEST1 accounted for 57% of disease cases. Further mutations were identified in CDHR1, GUCY2D, PROM1, CRX, GUCA1A, CERKL, MT-TL1, KIF11, RP1L1, MERTK, RDH5, CDH3, C1QTNF5, CRB1, JAG1, DRAM2, POC1B, NPHP1 and RPGR. We provide detailed illustrations of rare phenotypes, including autofluorescence and optical coherence tomography imaging. Targeted NGS also identified six potential novel genotype-phenotype correlations for FAM161A, INPP5E, MERTK, FBLN5, SEMA4A and IMPDH1. Clinical reassessment of genetically unsolved patients revealed subgroups with similar retinal phenotype, indicating a common molecular disease cause in each subgroup.

A family harboring homozygous FZD4 deletion supports the existence of recessive FZD4-related familial exudative vitreoretinopathy.

PURPOSE: To document recessive FZD4-related familial exudative vitreoretinopathy. METHODS: Retrospective case series. RESULTS: Two brothers, the only two males among five siblings, had bilateral infantile retinal detachments and were referred for genetic counseling. Next-generation sequencing uncovered a homozygous FZD4 frameshift deletion in both affected brothers (c.40_49delCCCGGGGGCG; p.Pro14Serfs*44). None of the other immediate family members had clinical evidence for retinal disease, including the three family members who underwent confirmatory genetic testing and were found to be heterozygous for the mutation (both parents and one sister). CONCLUSIONS: The findings in this family support the concept that some mutated FZD4 alleles can be associated with recessive rather than dominant disease.

[Xanthinuria type 1 in a woman with arthralgias: a combined clinical and molecular genetic investigation]. / Xanthinurie Typ 1 bei einer Patientin mit Gelenkschmerzen: Eine kombinierte konventionelle und molekulargenetische Ursachensuche.

HISTORY AND CLINICAL PRESENTATION: A 53-year old woman with recurrent polyarthralgias, negative test results in a recent rheumatologic work-up and an unmeasurably low uric acid serum concentration presented for suspected IgM paraproteinemia. INVESTIGATIONS: Physical examination, abdominal ultrasound and routine laboratory test results were unremarkable. Repeat determination confirmed a markedly decreased uric acid (UA) serum concentration. Urinary xanthine and hypoxanthine concentrations were increased by 14-fold and 7.5-fold, respectively. Fractional urinary UA excretion was not increased and the allopurinol loading test was normal. Sequencing of the xanthine dehydrogenase (XDH) gene revealed the pathogenic deletion c.641delC in the homozygous state. Segregation analysis showed that the patient's mother and her two adult sons were carriers of the mutation but not a half-sister and a half-brother of her deceased father. There was no evidence of parental consanguinity. These results established xanthinuria type 1 as the cause of the patient's recurrent polyarthralgias due to a previously unreported homozygosity for the known mutation c.641delC of the XDH gene. TREATMENT AND COURSE: The patient was advised to adhere to a low-purine diet and to ensure an increased daily fluid-intake of at least 2.5 l. She has since remained symptom free. CONCLUSION: Markedly lowered serum uric acid concentrations are a hallmark of xanthinuria and of hereditary renal hypouricemia, and in the absence of severe hepatic failure or evidence of an untoward drug effect should raise suspicion of these diseases. A targeted diagnostic work-up should then be initiated and factitious hypouricemia due to IgM paraproteinemia considered only in the case of equivocal test results. Molecular-genetic characterization and segregation analysis will ultimately establish the underlying genotype.

CCDC22 deficiency in humans blunts activation of proinflammatory NF-κB signaling.

NF-κB is a master regulator of inflammation and has been implicated in the pathogenesis of immune disorders and cancer. Its regulation involves a variety of steps, including the controlled degradation of inhibitory IκB proteins. In addition, the inactivation of DNA-bound NF-κB is essential for its regulation. This step requires a factor known as copper metabolism Murr1 domain-containing 1 (COMMD1), the prototype member of a conserved gene family. While COMMD proteins have been linked to the ubiquitination pathway, little else is known about other family members. Here we demonstrate that all COMMD proteins bind to CCDC22, a factor recently implicated in X-linked intellectual disability (XLID). We showed that an XLID-associated CCDC22 mutation decreased CCDC22 protein expression and impaired its binding to COMMD proteins. Moreover, some affected individuals displayed ectodermal dysplasia, a congenital condition that can result from developmental NF-κB blockade. Indeed, patient-derived cells demonstrated impaired NF-κB activation due to decreased IκB ubiquitination and degradation. In addition, we found that COMMD8 acted in conjunction with CCDC22 to direct the degradation of IκB proteins. Taken together, our results indicate that CCDC22 participates in NF-κB activation and that its deficiency leads to decreased IκB turnover in humans, highlighting an important regulatory component of this pathway.

MCT8 mutation analysis and identification of the first female with Allan-Herndon-Dudley syndrome due to loss of MCT8 expression.

Mutations in the thyroid monocarboxylate transporter 8 gene (MCT8/SLC16A2) have been reported to result in X-linked mental retardation (XLMR) in patients with clinical features of the Allan-Herndon-Dudley syndrome (AHDS). We performed MCT8 mutation analysis including 13 XLMR families with LOD scores >2.0, 401 male MR sibships and 47 sporadic male patients with AHDS-like clinical features. One nonsense mutation (c.629insA) and two missense changes (c.1A>T and c.1673G>A) were identified. Consistent with previous reports on MCT8 missense changes, the patient with c.1673G>A showed elevated serum T3 level. The c.1A>T change in another patient affects a putative translation start codon, but the same change was present in his healthy brother. In addition normal serum T3 levels were present, suggesting that the c.1A>T (NM_006517) variation is not responsible for the MR phenotype but indicates that MCT8 translation likely starts with a methionine at position p.75. Moreover, we characterized a de novo translocation t(X;9)(q13.2;p24) in a female patient with full blown AHDS clinical features including elevated serum T3 levels. The MCT8 gene was disrupted at the X-breakpoint. A complete loss of MCT8 expression was observed in a fibroblast cell-line derived from this patient because of unfavorable nonrandom X-inactivation. Taken together, these data indicate that MCT8 mutations are not common in non-AHDS MR patients yet they support that elevated serum T3 levels can be indicative for AHDS and that AHDS clinical features can be present in female MCT8 mutation carriers whenever there is unfavorable nonrandom X-inactivation.

Mutation frequencies of X-linked mental retardation genes in families from the EuroMRX consortium.

The EuroMRX family cohort consists of about 400 families with non-syndromic and 200 families with syndromic X-linked mental retardation (XLMR). After exclusion of Fragile X (Fra X) syndrome, probands from these families were tested for mutations in the coding sequence of 90 known and candidate XLMR genes. In total, 73 causative mutations were identified in 21 genes. For 42% of the families with obligate female carriers, the mental retardation phenotype could be explained by a mutation. There was no difference between families with (lod score >2) or without (lod score <2) significant linkage to the X chromosome. For families with two to five affected brothers (brother pair=BP families) only 17% of the MR could be explained. This is significantly lower (P=0.0067) than in families with obligate carrier females and indicates that the MR in about 40% (17/42) of the BP families is due to a single genetic defect on the X chromosome. The mutation frequency of XLMR genes in BP families is lower than can be expected on basis of the male to female ratio of patients with MR or observed recurrence risks. This might be explained by genetic risk factors on the X chromosome, resulting in a more complex etiology in a substantial portion of XLMR patients. The EuroMRX effort is the first attempt to unravel the molecular basis of cognitive dysfunction by large-scale approaches in a large patient cohort. Our results show that it is now possible to identify 42% of the genetic defects in non-syndromic and syndromic XLMR families with obligate female carriers.

X-linked mental retardation: a comprehensive molecular screen of 47 candidate genes from a 7.4 Mb interval in Xp11.

About 30% of the mutations causing nonsyndromic X-linked mental retardation (MRX) are thought to be located in Xp11 and in the pericentromeric region, with a particular clustering of gene defects in a 7.4 Mb interval flanked by the genes ELK1 and ALAS2. To search for these mutations, 47 brain-expressed candidate genes located in this interval have been screened for mutations in up to 22 mental retardation (MR) families linked to this region. In total, we have identified 57 sequence variants in exons and splice sites of 27 genes. Based on these data, four novel MR genes were identified, but most of the sequence variants observed during this study have not yet been described. The purpose of this article is to present a comprehensive overview of this work and its outcome. It describes all sequence variants detected in 548 exons and their flanking sequences, including disease-causing mutations as well as possibly relevant polymorphic and silent sequence changes. We show that many of the studied genes are unlikely to play a major role in MRX. This information will help to avoid duplication of efforts in the ongoing endeavor to unravel the molecular causes of MRX.

Homozygosity mapping in consanguineous families reveals extreme heterogeneity of non-syndromic autosomal recessive mental retardation and identifies 8 novel gene loci.

Autosomal recessive gene defects are arguably the most important, but least studied genetic causes of severe cognitive dysfunction. Homozygosity mapping in 78 consanguineous Iranian families with nonsyndromic autosomal recessive mental retardation (NS-ARMR) has enabled us to determine the chromosomal localization of at least 8 novel gene loci for this condition. Our data suggest that in the Iranian population NS-ARMR is very heterogeneous, and they argue against the existence of frequent gene defects that account for more than a few percent of the cases.

A novel X-linked recessive mental retardation syndrome comprising macrocephaly and ciliary dysfunction is allelic to oral-facial-digital type I syndrome.

We report on a large family in which a novel X-linked recessive mental retardation (XLMR) syndrome comprising macrocephaly and ciliary dysfunction co-segregates with a frameshift mutation in the OFD1 gene. Mutations of OFD1 have been associated with oral-facial-digital type 1 syndrome (OFD1S) that is characterized by X-chromosomal dominant inheritance and lethality in males. In contrast, the carrier females of our family were clinically inconspicuous, and the affected males suffered from severe mental retardation, recurrent respiratory tract infections and macrocephaly. All but one of the affected males died from respiratory problems in infancy; and impaired ciliary motility was confirmed in the index patient by high-speed video microscopy examination of nasal epithelium. This family broadens the phenotypic spectrum of OFD1 mutations in an unexpected way and sheds light on the complexity of the underlying disease mechanisms.

Novel JARID1C/SMCX mutations in patients with X-linked mental retardation.

X-linked mental retardation (XLMR) is a heterogeneous disorder that affects approximately 2 in 1000 males. JARID1C/SMCX is relatively new among the known XLMR genes, and seven different mutations have been identified previously in this gene [Jensen LR et al., Am. J. Hum. Genet. 76:227-236, 2005]. Here, we report five novel JARID1C mutations in five XLMR families. The changes comprise one nonsense mutation (p.Arg332X) and four missense mutations (p.Asp87Gly; p.Phe642Leu; p.Arg750Trp; p.Tyr751Cys) affecting evolutionarily conserved amino acids. The degree of mental retardation in the affected males ranged from mild to severe, and some patients suffered from additional disorders such as epilepsy, short stature, or behavioral problems. This study brings the total number of reported JARID1C mutations to twelve. In contrast to other XLMR genes in which mutations were found only in single or very few families, JARID1C appears to be one of the more frequently mutated genes in this disorder.

SNP array-based homozygosity mapping reveals MCPH1 deletion in family with autosomal recessive mental retardation and mild microcephaly.

Very little is known about the molecular basis of autosomal recessive MR (ARMR) because in developed countries, small family sizes preclude mapping and identification of the relevant gene defects. We therefore chose to investigate genetic causes of ARMR in large consanguineous Iranian families. This study reports on a family with six mentally retarded members. Array-based homozygosity mapping and high-resolution microarray-based comparative genomic hybridization (array CGH) revealed a deletion of approximately 150-200 kb, encompassing the promoter and the first six exons of the MCPH1 gene, one out of four genes that have been previously implicated in ARMR with microcephaly. Reexamination of affected individuals revealed a high proportion of prematurely condensed chromosomes, which is a hallmark of this condition, but in spite of the severity of the mutation, all patients showed only borderline to mild microcephaly. Therefore the phenotypic spectrum of MCPH1 mutations may be wider than previously assumed, with ARMR being the only consistent clinical finding.

Voltage-dependent ion channels in the mouse RPE: comparison with Norrie disease mice.

We studied electrophysiological properties of cultured retinal pigment epithelial (RPE) cells from mouse and a mouse model for Norrie disease. Wild-type RPE cells revealed the expression of ion channels known from other species: delayed-rectifier K(+) channels composed of Kv1.3 subunits, inward rectifier K(+) channels, Ca(V)1.3 L-type Ca(2+) channels and outwardly rectifying Cl(-) channels. Expression pattern and the ion channel characteristics current density, blocker sensitivity, kinetics and voltage-dependence were compared in cells from wild-type and Norrie mice. Although no significant differences were observed, our study provides a base for future studies on ion channel function and dysfunction in transgenic mouse models.

CGHPRO -- a comprehensive data analysis tool for array CGH.

BACKGROUND: Array CGH (Comparative Genomic Hybridisation) is a molecular cytogenetic technique for the genome wide detection of chromosomal imbalances. It is based on the co-hybridisation of differentially labelled test and reference DNA onto arrays of genomic BAC clones, cDNAs or oligonucleotides, and after correction for various intervening variables, loss or gain in the test DNA can be indicated from spots showing aberrant signal intensity ratios. Now that this technique is no longer confined to highly specialized laboratories and is entering the realm of clinical application, there is a need for a user-friendly software package that facilitates estimates of DNA dosage from raw signal intensities obtained by array CGH experiments, and which does not depend on a sophisticated computational environment. RESULTS: We have developed a user-friendly and versatile tool for the normalization, visualization, breakpoint detection and comparative analysis of array-CGH data. CGHPRO is a stand-alone JAVA application that guides the user through the whole process of data analysis. The import option for image analysis data covers several data formats, but users can also customize their own data formats. Several graphical representation tools assist in the selection of the appropriate normalization method. Intensity ratios of each clone can be plotted in a size-dependent manner along the chromosome ideograms. The interactive graphical interface offers the chance to explore the characteristics of each clone, such as the involvement of the clones sequence in segmental duplications. Circular Binary Segmentation and unsupervised Hidden Markov Model algorithms facilitate objective detection of chromosomal breakpoints. The storage of all essential data in a back-end database allows the simultaneously comparative analysis of different cases. The various display options facilitate also the definition of shortest regions of overlap and simplify the identification of odd clones. CONCLUSION: CGHPRO is a comprehensive and easy-to-use data analysis tool for array CGH. Since all of its features are available offline, CGHPRO may be especially suitable in situations where protection of sensitive patient data is an issue. It is distributed under GNU GPL licence and runs on Linux and Windows.

Mutations in the JARID1C gene, which is involved in transcriptional regulation and chromatin remodeling, cause X-linked mental retardation.

In families with nonsyndromic X-linked mental retardation (NS-XLMR), >30% of mutations seem to cluster on proximal Xp and in the pericentric region. In a systematic screen of brain-expressed genes from this region in 210 families with XLMR, we identified seven different mutations in JARID1C, including one frameshift mutation and two nonsense mutations that introduce premature stop codons, as well as four missense mutations that alter evolutionarily conserved amino acids. In two of these families, expression studies revealed the almost complete absence of the mutated JARID1C transcript, suggesting that the phenotype in these families results from functional loss of the JARID1C protein. JARID1C (Jumonji AT-rich interactive domain 1C), formerly known as "SMCX," is highly similar to the Y-chromosomal gene JARID1D/SMCY, which encodes the H-Y antigen. The JARID1C protein belongs to the highly conserved ARID protein family. It contains several DNA-binding motifs that link it to transcriptional regulation and chromatin remodeling, processes that are defective in various other forms of mental retardation. Our results suggest that JARID1C mutations are a relatively common cause of XLMR and that this gene might play an important role in human brain function.

Mutations in the FTSJ1 gene coding for a novel S-adenosylmethionine-binding protein cause nonsyndromic X-linked mental retardation.

Nonsyndromic X-linked mental retardation (NSXLMR) is a very heterogeneous condition, and most of the underlying gene defects are still unknown. Recently, we have shown that approximately 30% of these genes cluster on the proximal Xp, which prompted us to perform systematic mutation screening in brain-expressed genes from this region. Here, we report on a novel NSXLMR gene, FTSJ1, which harbors mutations in three unrelated families--one with a splicing defect, one with a nonsense mutation, and one with a deletion of one nucleotide. In two families, subsequent expression studies showed complete absence or significant reduction of mutant FTSJ1 transcripts. FTSJ1 protein is a homolog of Escherichia coli RNA methyltransferase FtsJ/RrmJ and may play a role in the regulation of translation. Further studies aim to elucidate the function of human FTSJ1 and its role during brain development.

Mutations in the polyglutamine binding protein 1 gene cause X-linked mental retardation.

We found mutations in the gene PQBP1 in 5 of 29 families with nonsyndromic (MRX) and syndromic (MRXS) forms of X-linked mental retardation (XLMR). Clinical features in affected males include mental retardation, microcephaly, short stature, spastic paraplegia and midline defects. PQBP1 has previously been implicated in the pathogenesis of polyglutamine expansion diseases. Our findings link this gene to XLMR and shed more light on the pathogenesis of this common disorder.

Duplication of the MID1 first exon in a patient with Opitz G/BBB syndrome.

Opitz G/BBB syndrome is a malformation syndrome of the ventral midline mainly characterized by hypertelorism, swallowing difficulties, hypospadias and developmental delay. SSCP analysis and genomic sequencing of the MID1 open reading frame have identified mutations in 80% of the families with X-linked inheritance. However, in many patients the underlying genetic defect remains undetected by these techniques. Using RNA diagnostics we have now identified a duplication of the MID1 first exon in a patient with X-linked Opitz G/BBB syndrome. This duplication introduces a premature termination codon. In addition, we could significantly lower the threshold for mutation detection on the DNA level by combining SSCP analysis with DHPLC technology.