Genetic evaluation of patients with Alström syndrome in the Polish population

Alström syndrome (AS) is a rare syndromic form of obesity and type 2 diabetes (T2D) in children coexisting with retinal dystrophy and disorders of many organs caused by the mutations in ALMS1 gene. Aim of this study was to identify the causative mutations in ALMS1 in a group of 12 patients of Polish origin with clinical symptoms of AS, and their 21 first‐degree relatives. Using DNA sequencing, nine different mutations including three novel were identified. These mutations were not present in 212 Polish individuals with no symptoms of AS, subjected to whole‐exome sequencing and collected in a national registry. Looking for genotype–phenotype relationships, we confirmed a severe phenotype in a boy with homozygous mutation in exon 16, and a relationship between a presence of T2D and mutations in exon 19. Evaluation of the type of mutation and its clinical effects gives hope for earlier diagnosis of AS in future patients and more advanced therapeutic approaches for patients with already diagnosed AS.     

Alström Syndrome: Mutation Spectrum of ALMS1

Alström Syndrome (ALMS), a recessive, monogenic ciliopathy caused by mutations in ALMS1, is typically characterized by multisystem involvement including early cone‐rod retinal dystrophy and blindness, hearing loss, childhood obesity, type 2 diabetes mellitus, cardiomyopathy, fibrosis, and multiple organ failure. The precise function of ALMS1 remains elusive, but roles in endosomal and ciliary transport and cell cycle regulation have been shown. The aim of our study was to further define the spectrum of ALMS1 mutations in patients with clinical features of ALMS. Mutational analysis in a world‐wide cohort of 204 families identified 109 novel mutations, extending the number of known ALMS1 mutations to 239 and highlighting the allelic heterogeneity of this disorder. This study represents the most comprehensive mutation analysis in patients with ALMS, identifying the largest number of novel mutations in a single study worldwide. Here, we also provide an overview of all ALMS1 mutations identified to date.     

Molecular analysis and long-term clinical evaluation of three siblings with Alström syndrome

Alström syndrome is a rare, autosomal recessive disorder characterized by a wide spectrum of clinical features including early‐onset retinal degeneration leading to blindness, sensorineural hearing loss, short stature, obesity, type 2 diabetes, hyperlipidemia and dilated cardiomyopathy. Renal, hepatic and pulmonary dysfunction may occur in the later phases of the disease. The three affected sisters, from a consanguineous Turkish family, with the characteristic features of Alström syndrome, were clinically diagnosed in 1987 and followed for 20 years. DNA sequence analysis of ALMS1, the causative gene in Alström syndrome, identified a novel homozygous disease‐causing mutation, c.8164C>T, resulting in a premature termination codon in exon 10 in each of the three affected sisters. Furthermore, we describe the longitudinal disease progression in this family and report new clinical findings likely associated with Alström syndrome, such as pes planus and hyperthyroidism.     

Atypical Alstrom syndrome with novel ALMS1 mutations precluded by current diagnostic criteria

We report on clinical and genetic studies in a non-consanguineous Irish sib-pair with infantile dilated cardiomyopathy and retinopathy. A diagnosis of Alström Syndrome (AS) was considered and diagnostic testing pursued. The Alströms gene (ALMS1) is very large (23 exons) and diagnostic testing of mutational hotspots (exon 6, 8 and 10) was negative. Furthermore the siblings were tall and did not have the typical phenotype of nystagmus, photophobia, obesity or hearing loss and so the AS diagnosis was removed. We then sought to identify the causative gene in this family using whole exome sequencing. Unexpectedly, the exome analysis identified novel compound heterozygous ALMS1 mutations in exon 5 (c.777delT:p.D260fs*26) and exon 20 (c.12145_12146insC:p.S4049fs*36) that segregated with the phenotype. Although the siblings show some clinical overlap with AS, their phenotype is not classical. It is plausible that their atypical presentation may be due to the location of the ALMS1 mutations outside the usual mutational hotspots. Our findings show how atypical cases of AS may be missed under the current diagnostic guidelines and support consideration of complete ALMS1 sequencing in children with two or more features, even if all of the core clinical features of AS are not present.     

Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290‐related phenotypes

Leber Congenital Amaurosis (LCA), the most severe inherited retinal dystrophy, is genetically heterogeneous, with 14 genes accounting for 70% of patients. Here, 91 LCA probands underwent LCA chip analysis and subsequent sequencing of 6 genes (CEP290, CRB1, RPE65, GUCY2D, AIPL1and CRX), revealing mutations in 69% of the cohort, with major involvement of CEP290 (30%). In addition, 11 patients with early‐onset retinal dystrophy (EORD) and 13 patients with Senior‐Loken syndrome (SLS), LCA‐Joubert syndrome (LCA‐JS) or cerebello‐oculo‐renal syndrome (CORS) were included. Exhaustive re‐inspection of the overall phenotypes in our LCA cohort revealed novel insights mainly regarding the CEP290‐related phenotype. The AHI1 gene was screened as a candidate modifier gene in three patients with the same CEP290 genotype but different neurological involvement. Interestingly, a heterozygous novel AHI1 mutation, p.Asn811Lys, was found in the most severely affected patient. Moreover, AHI1 screening in five other patients with CEP290‐related disease and neurological involvement revealed a second novel missense variant, p.His758Pro, in one LCA patient with mild mental retardation and autism. These two AHI1 mutations might thus represent neurological modifiers of CEP290‐related disease. © 2010 Wiley‐Liss, Inc.     

Homozygous loss-of-function mutation in ALMS1 causes the lethal disorder mitogenic cardiomyopathy in two siblings

BackgroundTwo siblings from consanguineous parents of Turkish descent presented with isolated dilated cardiomyopathy, leading to early death in infancy. The diagnosis of mitogenic cardiomyopathy was made histologically.Methods and resultsLinkage analysis combined with exome sequencing identified a homozygous deleterious mutation in the ALMS1 gene as the cause of this phenotype.ConclusionsAlström syndrome is characterized by a typically transient dilating cardiomyopathy in infancy, suggesting that mitogenic cardiomyopathy represents the extreme phenotype, resulting in demise before the other clinical symptoms become evident. This observation further illustrates the role of ALMS1 and cell cycle regulation.     

Whole exome sequencing identifies multiple diagnoses in congenital glaucoma with systemic anomalies

The genetic basis of congenital glaucoma with systemic anomalies is largely unknown. Whole exome sequencing (WES) in 10 probands with congenital glaucoma and variable systemic anomalies identified pathogenic or likely pathogenic variants in three probands; in two of these, a combination of two Mendelian disorders was found to completely explain the patients' features whereas in the third case only the ocular findings could be explained by the genetic diagnosis. The molecular diagnosis for glaucoma included two cases with compound heterozygous or homozygous pathogenic alleles in CYP1B1 and one family with a dominant pathogenic variant in FOXC1; the second genetic diagnosis for the additional systemic features included compound heterozygous mutations in NPHS1 in one family and a heterozygous 18q23 deletion in another pedigree. These findings show the power of WES in the analysis of complex conditions and emphasize the importance of CYP1B1 screening in patients with congenital glaucoma regardless of the presence/absence of other systemic anomalies.     

Mutations of MYH14 are associated to anorectal malformations with recto‐perineal fistulas in a small subset of Chinese population

Background: Anorectal malformations (ARMs) are among the most commonly congenital abnormalities of distal hindgut development, ranging from anal stenosis to anal atresia with or without fistulas and persistent cloaca. The etiology remains elusive for most ARM cases and the majority of genetic studies on ARMs were based on a candidate gene approach. Materials and Methods: In all eight family members of a non‐consanguineous Chinese family, we performed whole‐exome sequencing. Subsequently, exome sequencing of MYH14 in 72 unrelated probands with ARMs was performed. The accurate distribution of non‐muscle myosin II heavy chain (NMHC II) was investigated by immunohistochemistry in serial sagittal sections of E11.5–13.5 mouse cloacal regions. Results: A homozygous mutation in MYH14 was identified in the two siblings of family 1. Compound heterozygous MYH14 changes were identified in an unrelated individual. Immunohistochemical analysis suggest stronger NMHC IIC localization in the epithelium of the murine embryonic cloaca, urorectal septum and hindgut compared with another two NMHC II isoforms. Conclusion: This is the first identification of mutations in MYH14 as a cause of ARMs. The stronger localization of NMHC IIC in E11.5–13.5 mouse cloacal regions further supports the role of MYH14 in anorectal development.     

Genome‐wide linkage and sequence analysis challenge CCDC66 as a human retinal dystrophy candidate gene and support a distinct NMNAT1‐related fundus phenotype

To uncover the genotype underlying early‐onset cone‐rod dystrophy and central nummular macular atrophic lesion in 2 siblings from an endogamous Arab family, we performed targeted next‐generation sequencing (NGS) of 44 retinal dystrophy genes, whole‐exome sequencing (WES) and genome‐wide linkage analysis. Targeted NGS and WES in the index patient highlighted 2 homozygous variants, a CCDC66 frameshift deletion and a novel missense NMNAT1 variant, c.500G>A (p.Asn167Ser). Linkage and segregation analysis excluded the CCDC66 variant and confirmed the NMNAT1 mutation. Biallelic NMNAT1 mutations cause Leber congenital amaurosis with a central nummular macular atrophic lesion (LCA9). The NMNAT1 mutation reported here underlied cone‐rod dystrophy rather than LCA but the fundus lesion was compatible with that of LCA9 patients, highlighting that such a fundus appearance should raise suspicion for biallelic mutations in NMNAT1 when in the context of any retinal dystrophy. Although Ccdc66 mutations have been proposed to cause retinal disease in dogs, our results and public databases challenge CCDC66 as a candidate gene for human retinal dystrophy.     

Monogenic diabetes syndromes: Locus‐specific databases for Alström,Wolfram, and Thiamine‐responsive megaloblastic anemia

We developed a variant database for diabetes syndrome genes, using the Leiden Open Variation Database platform, containing observed phenotypes matched to the genetic variations. We populated it with 628 published disease‐associated variants (December 2016) for: WFS1 (n = 309), CISD2 (n = 3), ALMS1 (n = 268), and SLC19A2 (n = 48) for Wolfram type 1, Wolfram type 2, Alström, and Thiamine‐responsive megaloblastic anemia syndromes, respectively; and included 23 previously unpublished novel germline variants in WFS1 and 17 variants in ALMS1. We then investigated genotype–phenotype relations for the WFS1 gene. The presence of biallelic loss‐of‐function variants predicted Wolfram syndrome defined by insulin‐dependent diabetes and optic atrophy, with a sensitivity of 79% (95% CI 75%–83%) and specificity of 92% (83%–97%). The presence of minor loss‐of‐function variants in WFS1 predicted isolated diabetes, isolated deafness, or isolated congenital cataracts without development of the full syndrome (sensitivity 100% [93%–100%]; specificity 78% [73%–82%]). The ability to provide a prognostic prediction based on genotype will lead to improvements in patient care and counseling. The development of the database as a repository for monogenic diabetes gene variants will allow prognostic predictions for other diabetes syndromes as next‐generation sequencing expands the repertoire of genotypes and phenotypes. The database is publicly available online at https://lovd.euro-wabb.org .     

Screening of a Large Cohort of Leber Congenital Amaurosis and Retinitis Pigmentosa Patients Identifies Novel LCA5 Mutations and New Genotype–Phenotype Correlations

This study was undertaken to investigate the prevalence of sequence variants in LCA5 in patients with Leber congenital amaurosis (LCA), early‐onset retinal dystrophy (EORD), and autosomal recessive retinitis pigmentosa (arRP); to delineate the ocular phenotypes; and to provide an overview of all published LCA5 variants in an online database. Patients underwent standard ophthalmic evaluations after providing informed consent. In selected patients, optical coherence tomography (OCT) and fundus autofluorescence imaging were possible. DNA samples from 797 unrelated patients with LCA and 211 with the various types of retinitis pigmentosa (RP) were screened by Sanger sequence analysis of all LCA5 exons and intron/exon junctions. Some LCA patients were prescreened by APEX technology or selected based on homozygosity mapping. In silico analyses were performed to assess the pathogenicity of the variants. Segregation analysis was performed where possible. Published and novel LCA5 variants were collected, amended for their correct nomenclature, and listed in a Leiden Open Variation Database (LOVD). Sequence analysis identified 18 new probands with 19 different LCA5 variants. Seventeen of the 19 LCA5 variants were novel. Except for two missense variants and one splice site variant, all variants were protein‐truncating mutations. Most patients expressed a severe phenotype, typical of LCA. However, some LCA subjects had better vision and intact inner segment/outer segment (IS/OS) junctions on OCT imaging. In two families with LCA5 variants, the phenotype was more compatible with EORD with affected individuals displaying preserved islands of retinal pigment epithelium. One of the families with a milder phenotype harbored a homozygous splice site mutation; a second family was found to have a combination of a stop mutation and a missense mutation. This is the largest LCA5 study to date. We sequenced 1,008 patients (797 with LCA, 211 with arRP) and identified 18 probands with LCA5 mutations. Mutations in LCA5 are a rare cause of childhood retinal dystrophy accounting for ～2% of disease in this cohort, and the majority of LCA5 mutations are likely null. The LCA5 protein truncating mutations are predominantly associated with LCA. However, in two families with the milder EORD, the LCA5 gene analysis revealed a homozygous splice site mutation in one and a stop mutation in combination with a missense mutation in a second family, suggesting that this milder phenotype is due to residual function of lebercilin and expanding the currently known phenotypic spectrum to include the milder early onset RP. Some patients have remaining foveal cone structures (intact IS/OS junctions on OCT imaging) and remaining visual acuities, which may bode well for upcoming treatment trials.     

Hidden Genetic Variation in LCA9‐Associated Congenital Blindness Explained by 5′UTR Mutations and Copy‐Number Variations of NMNAT1

Leber congenital amaurosis (LCA) is a severe autosomal‐recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9‐associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5′UTR variant, c.‐70A>T. Moreover, an adjacent 5′UTR variant, c.‐69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5′UTR variants resulted in decreased NMNAT1 mRNA abundance in patients’ lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE‐1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu‐rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1‐associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat‐mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness.     

Next‐generation DNA sequencing of a Swedish malignant hyperthermia cohort

Malignant hyperthermia (MH)‐related mutations have been identified in the ryanodine receptor type 1 gene (RYR1) and in the dihydropyridine gene (CACNA1S), but about half of the patients do not have causative mutations in these genes. We wanted to study the contribution of other muscle genes to the RYR1 phenotypes. We designed a gene panel for sequence enrichment targeting 64 genes of proteins involved in the homeostasis of the striated muscle cell. Next‐generation sequencing (NGS) resulted in >50,000 sequence variants which were further analyzed by software filtering criteria to identify causative variants. In four of five patients we identified previously reported RYR1 mutations while the fifth patient did not show any candidate variant in any of the genes investigated. In two patients pathogenic variants were found in other genes known to cause a muscle disorders. All but one patient carried likely benign rare polymorphisms. The NGS technique proved convenient in identifying variants in the RYR1. However, with a clinically variable phenotype‐like MH, the pre‐selection of genes poses problems in variant interpretation.     

Dental and extra‐oral clinical features in 41 patients with WNT10A gene mutations: A multicentric genotype–phenotype study

WNT10A gene encodes a canonical wingless pathway signaling molecule involved in cell fate specification as well as morphogenetic patterning of the developing ectoderm, nervous system, skeleton, and tooth. In patients, WNT10A mutations are responsible for ectodermal‐derived pathologies including isolated hypo‐oligodontia, tricho‐odonto‐onycho‐dermal dysplasia and Schöpf‐Schulz‐Passarge syndrome (SSPS). Here we describe the dental, ectodermal, and extra‐ectodermal phenotypic features of a cohort of 41 patients from 32 unrelated families. Correlations with WNT10A molecular status (heterozygous carrier, compound heterozygous, homozygous) and patient's phenotypes were performed. Mild to severe oligodontia was observed in all patients bearing biallelic WNT10A mutations. However, patients with compound heterozygous mutations presented no significant difference in phenotypes compared with homozygous individuals. Anomalies in tooth morphology were frequently observed with heterozygous patients displaying hypodontia. No signs of SSPS, especially eyelids cysts, were detected in our cohort. Interestingly, extra‐ectodermal signs consisted of skeletal, neurological and vascular anomalies, the latter suggesting a wider phenotypic spectrum associated with WNT10A mutations. Indeed, the Wnt pathway plays a crucial role in skeletal development, lipid metabolism, and neurogenesis, potentially explaining patient's clinical manifestations.     

Collagen type IV‐related nephropathies in Portugal: pathogenic COL4A3 and COL4A4 mutations and clinical characterization of 25 families

Pathogenic mutations in genes COL4A3/COL4A4 are responsible for autosomal Alport syndrome (AS) and thin basement membrane nephropathy (TBMN). We used Sanger sequencing to analyze all exons and splice site regions of COL4A3/COL4A4, in 40 unrelated Portuguese probands with clinical suspicion of AS/TBMN. To assess genotype–phenotype correlations, we compared clinically relevant phenotypes/outcomes between homozygous/compound heterozygous and apparently heterozygous patients. Seventeen novel and four reportedly pathogenic COL4A3/COL4A4 mutations were identified in 62.5% (25/40) of the probands. Regardless of the mutated gene, all patients with ARAS manifested chronic renal failure (CRF) and hearing loss, whereas a minority of the apparently heterozygous patients had CRF or extrarenal symptoms. CRF was diagnosed at a significantly younger age in patients with ARAS. In our families, the occurrence of COL4A3/COL4A4 mutations was higher, while the prevalence of XLAS was lower than expected. Overall, a pathogenic COL4A3/COL4A4/COL4A5 mutation was identified in >50% of patients with fewer than three of the standard diagnostic criteria of AS. With such a population background, simultaneous next‐generation sequencing of all three genes may be recommended as the most expedite approach to diagnose collagen IV‐related glomerular basement membrane nephropathies.     

Role of ADAMTSL4 mutations in FBN1 mutation‐negative ectopia lentis patients

Ectopia lentis (EL) is genetically heterogeneous with both autosomal‐dominant and ‐recessive forms. The dominant disorder can be caused by mutations in FBN1, at the milder end of the type‐1 fibrillinopathies spectrum. Recently in a consanguineous Jordanian family, recessive EL was mapped to locus 1q21 containing the ADAMTSL4 gene and a nonsense mutation was found in exon 11 (c.1785T>G, p.Y595X). In this study, 36 consecutive probands with EL who did not fulfill the Ghent criteria for MFS were screened for mutations in FBN1 and ADAMTSL4. Causative FBN1 mutations were identified in 23/36 (64%) of probands while homozygous or compound heterozygous ADAMTSL4 mutations were identified in 6/12 (50%) of the remaining probands. Where available, familial screening of these families confirmed the mutation co‐segregated with the EL phenotype. This study confirms that homozygous mutations in ADAMTSL4 are associated with autosomal‐recessive EL in British families. Furthermore; the first compound heterozygous mutation is described resulting in a PTC and a missense mutation in the PLAC (protease and lacunin) domain. The identification of a causative mutation in ADAMTSL4 may allow the exclusion of Marfan syndrome in these families and guide the clinical management, of particular relevance in young children affected by EL. © 2010 Wiley‐Liss, Inc.     

Whole-exome sequencing identifies ALMS1, IQCB1, CNGA3, and MYO7A mutations in patients with Leber congenital amaurosis

It has been well documented that mutations in the same retinal disease gene can result in different clinical phenotypes due to difference in the mutant allele and/or genetic background. To evaluate this, a set of consanguineous patient families with Leber congenital amaurosis (LCA) that do not carry mutations in known LCA disease genes was characterized through homozygosity mapping followed by targeted exon/whole-exome sequencing to identify genetic variations. Among these families, a total of five putative disease-causing mutations, including four novel alleles, were found for six families. These five mutations are located in four genes, ALMS1, IQCB1, CNGA3, and MYO7A. Therefore, in our LCA collection from Saudi Arabia, three of the 37 unassigned families carry mutations in retinal disease genes ALMS1, CNGA3, and MYO7A, which have not been previously associated with LCA, and 3 of the 37 carry novel mutations in IQCB1, which has been recently associated with LCA. Together with other reports, our results emphasize that the molecular heterogeneity underlying LCA, and likely other retinal diseases, may be highly complex. Thus, to obtain accurate diagnosis and gain a complete picture of the disease, it is essential to sequence a larger set of retinal disease genes and combine the clinical phenotype with molecular diagnosis.     

A Next Generation Sequencing custom gene panel as first line diagnostic tool for atypical cases of syndromic obesity: Application in a case of Alström syndrome

Obesity phenotype can be manifested as an isolated trait or accompanied by multisystem disorders as part of a syndromic picture. In both situations, same molecular pathways may be involved to different degrees. This evidence is stronger in syndromic obesity, in which phenotypes of different syndromes may overlap. In these cases, genetic testing can unequivocally provide a final diagnosis. Here we describe a patient who met the diagnostic criteria for Alström syndrome only during adolescence. Genetic testing was requested at 25 years of age for a final confirmation of the diagnosis. The genetic diagnosis of Alström syndrome was obtained through a Next Generation Sequencing genetic test approach using a custom-designed gene panel of 47 genes associated with syndromic and non-syndromic obesity. Genetic analysis revealed a novel homozygous frameshift variant p.(Arg1550Lysfs*10) on exon 8 of the ALMS1 gene.This case shows the need for a revision of the diagnostic criteria guidelines, as a consequence of the recent advent of massive parallel sequencing technology. Indications for genetic testing reported in these currently accepted diagnostic criteria for Alström syndrome, were drafted when sequencing was expensive and time consuming. Nowadays, Next Generation Sequencing testing could be considered as first line diagnostic tool not only for Alström syndrome but, more generally, for all those atypical or not clearly distinguishable cases of syndromic obesity, thus avoiding delayed diagnosis and treatments. Early diagnosis permits a better follow-up and pre-symptomatic interventions.     

Mutation in The Nuclear‐Encoded Mitochondrial Isoleucyl–tRNA Synthetase IARS2 in Patients with Cataracts,Growth Hormone Deficiency with Short Stature,Partial Sensorineural Deafness,and Peripheral Neuropathy or with Leigh Syndrome

Mutations in the nuclear‐encoded mitochondrial aminoacyl–tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short‐stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole‐exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl–tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations.