Exome sequencing of 1190 non-syndromic clubfoot cases reveals HOXD12 as a novel disease gene.

BACKGROUND: Clubfoot, presenting as a rigid inward and downward turning of the foot, is one of the most common congenital musculoskeletal anomalies. The aetiology of clubfoot is poorly understood and variants in known clubfoot disease genes account for only a small portion of the heritability. METHODS: Exome sequence data were generated from 1190 non-syndromic clubfoot cases and their family members from multiple ethnicities. Ultra-rare variant burden analysis was performed comparing 857 unrelated clubfoot cases with European ancestry with two independent ethnicity-matched control groups (1043 in-house and 56 885 gnomAD controls). Additional variants in prioritised genes were identified in a larger cohort, including probands with non-European ancestry. Segregation analysis was performed in multiplex families when available. RESULTS: Rare variants in 29 genes were enriched in clubfoot cases, including PITX1 (a known clubfoot disease gene), HOXD12, COL12A1, COL9A3 and LMX1B. In addition, rare variants in posterior HOX genes (HOX9-13) were enriched overall in clubfoot cases. In total, variants in these genes were present in 8.4% (100/1190) of clubfoot cases with both European and non-European ancestry. Among these, 3 are de novo and 22 show variable penetrance, including 4 HOXD12 variants that segregate with clubfoot. CONCLUSION: We report HOXD12 as a novel clubfoot disease gene and demonstrate a phenotypic expansion of known disease genes (myopathy gene COL12A1, Ehlers-Danlos syndrome gene COL9A3 and nail-patella syndrome gene LMX1B) to include isolated clubfoot.

Genetic evidence for splicing-dependent structural and functional plasticity in CASK protein.

BACKGROUND: Pontocerebellar hypoplasia (PCH) may present with supratentorial phenotypes and is often accompanied by microcephaly. Damaging mutations in the X-linked gene CASK produce self-limiting microcephaly with PCH in females but are often lethal in males. CASK deficiency leads to early degeneration of cerebellar granule cells but its role in other regions of the brain remains uncertain. METHOD: We generated a conditional Cask knockout mice and deleted Cask ubiquitously after birth at different times. We examined the clinical features in several subjects with damaging mutations clustered in the central part of the CASK protein. We have performed phylogenetic analysis and RT-PCR to assess the splicing pattern within the same protein region and performed in silico structural analysis to examine the effect of splicing on the CASK's structure. RESULT: We demonstrate that deletion of murine Cask after adulthood does not affect survival but leads to cerebellar degeneration and ataxia over time. Intriguingly, damaging hemizygous CASK mutations in boys who display microcephaly and cerebral dysfunction but without PCH are known. These mutations are present in two vertebrate-specific CASK exons. These exons are subject to alternative splicing both in forebrain and hindbrain. Inclusion of these exons differentially affects the molecular structure and hence possibly the function/s of the CASK C-terminus. CONCLUSION: Loss of CASK function disproportionately affects the cerebellum. Clinical data, however, suggest that CASK may have additional vertebrate-specific function/s that play a role in the mammalian forebrain. Thus, CASK has an ancient function shared between invertebrates and vertebrates as well as novel vertebrate-specific function/s.

Arrhythmias and cardiac MRI associations in patients with established cardiac dystrophinopathy.

AIMS: Some patients with cardiac dystrophinopathy die suddenly. Whether such deaths are preventable by specific antiarrhythmic management or simply indicate heart failure overwhelming medical therapies is uncertain. The aim of this prospective, cohort study was to describe the occurrence and nature of cardiac arrhythmias recorded during prolonged continuous ECG rhythm surveillance in patients with established cardiac dystrophinopathy and relate them to abnormalities on cardiac MRI. METHODS AND RESULTS: A cohort of 10 patients (36.3 years; 3 female) with LVEF<40% due to Duchenne (3) or Becker muscular (4) dystrophy or Duchenne muscular dystrophy-gene carrying effects in females (3) were recruited, had cardiac MRI, ECG signal-averaging and ECG loop-recorder implants. All were on standard of care heart medications and none had prior history of arrhythmias.No deaths or brady arrhythmias occurred during median follow-up 30 months (range 13-35). Self-limiting episodes of asymptomatic tachyarrhythmia (range 1-29) were confirmed in 8 (80%) patients (ventricular only 2; ventricular and atrial 6). Higher ventricular arrhythmia burden correlated with extent of myocardial fibrosis (extracellular volume%, p=0.029; native T1, p=0.49; late gadolinium enhancement, p=0.49), but not with LVEF% (p=1.0) on MRI and atrial arrhythmias with left atrial dilatation. Features of VT episodes suggested various underlying arrhythmia mechanisms. CONCLUSIONS: The overall prevalence of arrhythmias was low. Even in such a small sample size, higher arrhythmia counts occurred in those with larger scar burden and greater ventricular volume, suggesting key roles for myocardial stretch as well as disease progression in arrhythmogenesis. These features overlap with the stage of left ventricular dysfunction when heart failure also becomes overt. The findings of this pilot study should help inform the design of a definitive study of specific antiarrhythmic management in dystrophinopathy. TRIAL REGISTRATION NUMBER: ISRCTN15622536.

Diagnosis and management in Rubinstein-Taybi syndrome: first international consensus statement.

Rubinstein-Taybi syndrome (RTS) is an archetypical genetic syndrome that is characterised by intellectual disability, well-defined facial features, distal limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in either of two genes (CREBBP, EP300) which encode for the proteins CBP and p300, which both have a function in transcription regulation and histone acetylation. As a group of international experts and national support groups dedicated to the syndrome, we realised that marked heterogeneity currently exists in clinical and molecular diagnostic approaches and care practices in various parts of the world. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria for types of RTS (RTS1: CREBBP; RTS2: EP300), molecular investigations, long-term management of various particular physical and behavioural issues and care planning. The recommendations as presented here will need to be evaluated for improvements to allow for continued optimisation of diagnostics and care.

Pathogenic variants affecting the TB5 domain of the fibrillin-1 protein: not only in geleophysic/acromicric dysplasias but also in Marfan syndrome.

BACKGROUND: Marfan syndrome (MFS) is a multisystem disease with a unique combination of skeletal, cardiovascular and ocular features. Geleophysic/acromicric dysplasias (GPHYSD/ACMICD), characterised by short stature and extremities, are described as 'the mirror image' of MFS. The numerous FBN1 pathogenic variants identified in MFS are located all along the gene and lead to the same final pathogenic sequence. Conversely, in GPHYSD/ACMICD, the 28 known heterozygous FBN1 pathogenic variants all affect exons 41-42 encoding TGFß-binding protein-like domain 5 (TB5). METHODS: Since 1996, more than 5000 consecutive probands have been referred nationwide to our laboratory for molecular diagnosis of suspected MFS. RESULTS: We identified five MFS probands carrying distinct heterozygous pathogenic in-frame variants affecting the TB5 domain of FBN1. The clinical data showed that the probands displayed a classical form of MFS. Strikingly, one missense variant affects an amino acid that was previously involved in GPHYSD. CONCLUSION: Surprisingly, pathogenic variants in the TB5 domain of FBN1 can lead to two opposite phenotypes: GPHYSD/ACMICD and MFS, suggesting the existence of different pathogenic sequences with the involvement of tissue specificity. Further functional studies are ongoing to determine the precise role of this domain in the physiopathology of each disease.

Clinical Characteristics of Diabetes in People with Mitochondrial DNA 3243A>G Mutation in Korea.

Maternally inherited diabetes and deafness (MIDD) is a rare mitochondrial disorder primarily resulting from m.3243A>G mutation. The clinical characteristics of MIDD exhibit significant heterogeneity. Our study aims to delineate these characteristics and determine the potential correlation with m.3243A>G heteroplasmy levels. This retrospective, descriptive study encompassed patients with confirmed m.3243A>G mutation and diabetes mellitus at Seoul National University Hospital. Our cohort comprises 40 patients with MIDD, with a mean age at study enrollment of 33.3±12.9 years and an average % of heteroplasmy of 30.0%± 14.6% in the peripheral blood. The most prevalent comorbidity was hearing loss (90%), followed by albuminuria (61%), seizure (38%), and stroke (33%). We observed a significant negative correlation between % of heteroplasmy and age at diabetes diagnosis. These clinical features can aid in the suspicion of MIDD and further consideration of genetic testing for m.3243A>G mutation.

Molecular diagnosis, clinical evaluation and phenotypic spectrum of Townes-Brocks syndrome: insights from a large Chinese hearing loss cohort.

BACKGROUND: Townes-Brocks syndrome (TBS) is a rare genetic disorder characterised by multiple malformations. Due to its phenotypic heterogeneity and rarity, diagnosis and recognition of TBS can be challenging and there has been a lack of investigation of patients with atypical TBS in large cohorts and delineation of their phenotypic characteristics. METHODS: We screened SALL1 and DACT1 variants using next-generation sequencing in the China Deafness Genetics Consortium (CDGC) cohort enrolling 20 666 unrelated hearing loss (HL) cases. Comprehensive clinical evaluations were conducted on seven members from a three-generation TBS family. Combining data from previously reported cases, we also provided a landscape of phenotypes and genotypes of patients with TBS. RESULTS: We identified five novel and two reported pathogenic/likely pathogenic (P/LP) SALL1 variants from seven families. Audiological features in patients differed in severity and binaural asymmetry. Moreover, previously undocumented malformations in the middle and inner ear were detected in one patient. By comprehensive clinical evaluations, we further provide evidence for the causal relationship between SALL1 variation and certain endocrine abnormalities. Penetrance analysis within familial contexts revealed incomplete penetrance among first-generation patients with TBS and a higher disease burden among their affected offspring. CONCLUSION: This study presents the first insight of genetic screening for patients with TBS in a large HL cohort. We broadened the phenotypic-genotypic spectrum of TBS and our results supported an underestimated prevalence of TBS. Due to the rarity and phenotypic heterogeneity of rare diseases, broader spectrum molecular tests, especially whole genome sequencing, can improve the situation of underdiagnosis and provide effective recommendations for clinical management.

De novo heterozygous missense variants in CELSR1 as cause of fetal pleural effusions and progressive fetal hydrops.

Fetal hydrops as detected by prenatal ultrasound usually carries a poor prognosis depending on the underlying aetiology. We describe the prenatal and postnatal clinical course of two unrelated female probands in whom de novo heterozygous missense variants in the planar cell polarity gene CELSR1 were detected using exome sequencing. Using several in vitro assays, we show that the CELSR1 p.(Cys1318Tyr) variant disrupted the subcellular localisation, affected cell-cell junction, impaired planar cell polarity signalling and lowered proliferation rate. These observations suggest that deleterious rare CELSR1 variants could be a possible cause of fetal hydrops.

Whole-exome sequencing reveals causative genetic variants for several overgrowth syndromes in molecularly negative Beckwith-Wiedemann spectrum.

Background Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder caused by (epi)genetic alterations at 11p15. Because approximately 20% of patients test negative via molecular testing of peripheral blood leukocytes, the concept of Beckwith-Wiedemann spectrum (BWSp) was established to encompass a broader cohort with diverse and overlapping phenotypes. The prevalence of other overgrowth syndromes concealed within molecularly negative BWSp remains unexplored.Methods We conducted whole-exome sequencing (WES) on 69 singleton patients exhibiting molecularly negative BWSp. Variants were confirmed by Sanger sequencing or quantitative genomic PCR. We compared BWSp scores and clinical features between groups with classical BWS (cBWS), atypical BWS or isolated lateralised overgrowth (aBWS+ILO) and overgrowth syndromes identified via WES.Results Ten patients, one classified as aBWS and nine as cBWS, showed causative gene variants for Simpson-Golabi-Behmel syndrome (five patients), Sotos syndrome (two), Imagawa-Matsumoto syndrome (one), glycosylphosphatidylinositol biosynthesis defect 11 (one) or 8q duplication/9p deletion (one). BWSp scores did not distinguish between cBWS and other overgrowth syndromes. Birth weight and height in other overgrowth syndromes were significantly larger than in aBWS+ILO and cBWS, with varying intergroup frequencies of clinical features.Conclusion Molecularly negative BWSp encapsulates other syndromes, and considering both WES and clinical features may facilitate accurate diagnosis.

Congenital anaemia associated with loss-of-function variants in DNA polymerase epsilon 1.

DNA polymerase epsilon (Pol ε), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol ε have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE, encoding Pol ε catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole-knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol ε defect in humans, additionally providing unique evidence linking Pol ε to haematopoiesis.

A novel leukocyte adhesion deficiency type III mutation manifests functional importance of the compact FERM domain in kindlin-3.

BACKGROUND: Leukocyte adhesion deficiency III (LAD-III) is a rare autosomal recessive syndrome characterized by functional deficiencies of platelets and leukocytes that occurs due to mutations in the FERMT3 gene encoding kindlin-3. Kindlin-3 is a FERM domain-containing adaptor protein that is essential in integrin activation. We have previously demonstrated that the FERM domain of kindlin-3 is structurally compact and plays an important role in supporting integrin activation in a mouse model. The impact of destabilizing the compact FERM domain in kindlin-3 on the development of LAD-III in humans remains uncertain. OBJECTIVES: To use primary cells from a patient with LAD-III to validate the role of the compact FERM domain in kindlin-3 function in platelets and leukocytes. METHODS: The patient is a 4-year-old girl who since infancy has displayed clinical features of LAD-III. Patient platelets and leukocytes were functionally analyzed, and structural analysis of the kindlin-3 variant was conducted. RESULTS: We identified a novel homozygous missense mutation in the FERMT3 (c.412G>A, p.E138K) FERM domain. Substantially reduced levels of kindlin-3 were detected in the proband's platelets and leukocytes. Functional evaluation verified that integrin αIIbß3-mediated platelet activation, spreading, and aggregation and ß2-integrin-mediated neutrophil adhesion and spreading were significantly compromised. Structural analysis revealed that this newly identified E138K substitution in kindlin-3 destabilizes the compacted FERM domain, resulting in poor expression of kindlin-3 in blood cells and subsequent LAD-III. CONCLUSION: We have identified a novel missense mutation and verified the functional significance of the compact kindlin-3 FERM domain in supporting integrin functions in platelets and leukocytes.

Whole exome sequencing of 491 individuals with inherited retinal diseases reveals a large spectrum of variants and identification of novel candidate genes.

BACKGROUND: Inherited retinal diseases (IRDs) include a range of vision loss conditions caused by variants in different genes. The clinical and genetic heterogeneity make identification of the genetic cause challenging. Here, a cohort of 491 unsolved cases from our cohort of Israeli and Palestinian families with IRDs underwent whole exome sequencing (WES), including detection of CNVs as well as single nucleotide variants (SNVs). METHODS: All participants underwent clinical examinations. Following WES on DNA samples by 3 billion, initial SNV analysis was performed by 3 billion and SNV and CNV analysis by Franklin Genoox. The CNVs indicated by the programme were confirmed by PCR followed by gel electrophoresis. RESULTS: WES of 491 IRD cases revealed the genetic cause of disease in 51% of cases, of which 11% were due wholly or in part to CNVs. In two cases, we clarified previously incorrect or unclear clinical diagnoses. This analysis also identified ESRRB and DNM1 as potential novel genes. CONCLUSION: This analysis is the most extensive one to include CNVs to examine IRD causing genes in the Israeli and Palestinian populations. It has allowed us to identify the causative variant of many patients with IRDs including ones with unclear diagnoses and potential novel genes.

Genetic variations of type 2 and type 3 von Willebrand diseases in Thailand.

AIMS: Von Willebrand disease (VWD) is an inherited haemostatic disorder with a wide range of bleeding phenotypes based on von Willebrand factor (VWF) levels. Multiple assays including VWF gene analysis are employed to correctly diagnose VWD and its subtypes. However, data on VWF mutations among Southeast Asian populations are lacking. We, therefore, aimed to explore genetic variations in Thai patients with type 2 and type 3 VWD by whole exome sequencing (WES). METHODS: In this multicentre study, Thai patients with type 2 and type 3 VWD, according to the definitions and VWF levels recommended by the international guidelines, were recruited. WES was performed using DNA extracted from peripheral blood in all cases. The novel variants were verified by Sanger sequencing. RESULTS: Fifteen patients (73% females; median age at diagnosis 3.0 years) with type 2 (n=12) and type 3 VWD (n=3) from 14 families were enrolled. All patients harboured at least one VWF variant. Six missense (p.Arg1374Cys, p.Arg1374His, p.Arg1399Cys, p.Arg1597Trp, p.Ser1613Pro, p.Pro1648Arg) and one splice-site (c.3379+1G>A) variants in the VWF gene were formerly described. Notably, six VWF variants, including three missense (p.Met814Ile, p.Trp856Cys, p.Pro2032Leu), one deletion (c.2251delG) and two splice-site (c.7729+4A>C, c.8115+2delT) mutations were novelly identified. Compound heterozygosity contributed to type 2 and type 3 VWD phenotypes in two and one patients, respectively. CONCLUSIONS: Type 2 and type 3 VWD in Thailand demonstrate the mutational variations among VWF exons/introns with several unique variants. The WES-based approach potentially provides helpful information to verify VWD diagnosis and facilitate genetic counselling in clinical practice.

Borjeson-Forssman-Lehmann Syndrome: Clinical Features and Diagnostic Challenges.

Borjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked recessive disorder resulting from mutations in the PHF6 gene. The syndrome is characterized by short stature, obesity, hypogonadism, hypotonia, intellectual disability, distinctive facial features, fleshy ears, and finger and toe abnormalities. However, the diagnostic challenge in identifying BFLS remains a topic of interest. In this case report, we present the clinical characteristics of a proband with BFLS, highlighting the additional features of hypotonia, intellectual disability, and distinctive facial features. While no definitive treatment exists for BFLS, patients benefit from specialized education and ongoing supervision from early childhood through adulthood. Symptomatic treatment, including close follow-up, may be necessary for complications such as seizures and hearing problems. Mastectomy or testosterone replacement therapy may be considered on a case-by-case basis. Genetic counseling for X-linkage should be offered to affected families.

Head-to-head trial of pegunigalsidase alfa versus agalsidase beta in patients with Fabry disease and deteriorating renal function: results from the 2-year randomised phase III BALANCE study.

BACKGROUND: Pegunigalsidase alfa is a PEGylated α-galactosidase A enzyme replacement therapy. BALANCE (NCT02795676) assessed non-inferiority of pegunigalsidase alfa versus agalsidase beta in adults with Fabry disease with an annualised estimated glomerular filtration rate (eGFR) slope more negative than -2 mL/min/1.73 m2/year who had received agalsidase beta for ≥1 year. METHODS: Patients were randomly assigned 2:1 to receive 1 mg/kg pegunigalsidase alfa or agalsidase beta every 2 weeks for 2 years. The primary efficacy analysis assessed non-inferiority based on median annualised eGFR slope differences between treatment arms. RESULTS: Seventy-seven patients received either pegunigalsidase alfa (n=52) or agalsidase beta (n=25). At baseline, mean (range) age was 44 (18-60) years, 47 (61%) patients were male, median eGFR was 74.5 mL/min/1.73 m2 and median (range) eGFR slope was -7.3 (-30.5, 6.3) mL/min/1.73 m2/year. At 2 years, the difference between median eGFR slopes was -0.36 mL/min/1.73 m2/year, meeting the prespecified non-inferiority margin. Minimal changes were observed in lyso-Gb3 concentrations in both treatment arms at 2 years. Proportions of patients experiencing treatment-related adverse events and mild or moderate infusion-related reactions were similar in both groups, yet exposure-adjusted rates were 3.6-fold and 7.8-fold higher, respectively, with agalsidase beta than pegunigalsidase alfa. At the end of the study, neutralising antibodies were detected in 7 out of 47 (15%) pegunigalsidase alfa-treated patients and 6 out of 23 (26%) agalsidase beta-treated patients. There were no deaths. CONCLUSIONS: Based on rate of eGFR decline over 2 years, pegunigalsidase alfa was non-inferior to agalsidase beta. Pegunigalsidase alfa had lower rates of treatment-emergent adverse events and mild or moderate infusion-related reactions. TRIAL REGISTRATION NUMBER: NCT02795676.

Carriers of autosomal recessive conditions: are they really 'unaffected?'

Mendel's Law of Dominance suggests that recessive disease expression requires the inheritance of two mutated alleles as the dominant, wildtype allele suppresses disease presentation leading to the expression of physiological normal phenotypes. However, there is existing evidence that challenges this school of thought. Here, we summarise existing literature evaluating metabolic and health impacts among carriers of autosomal recessive conditions, focusing on phenylketonuria (PKU), classical homocystinuria, galactosemia and Usher syndrome as examples. Our findings suggest that carriers, often described as 'unaffected', may actually display attenuated symptoms for the recessive disease they are carrying. For instance, PKU is an inborn error of metabolism characterised by the build-up of plasma phenylalanine attributed to the deficiency of the phenylalanine hydroxylase (PAH) enzyme. While less severe, PKU carriers also exhibit this impaired enzymatic activity, leading to elevated plasma phenylalanine levels, especially after phenylalanine consumption. Related to these metabolic alterations in the PAH pathway, there is early evidence to suggest that PKU carriers may have compromised cognitive and mental health outcomes. Overall, research on the health and metabolic impacts of PKU carriers is sparse, with most studies conducted several decades ago. However, early evidence suggests that intermediate phenotypes among carriers of autosomal recessive conditions are plausible. The illustrated possible intermediate phenotypes observed among carriers necessitates future research to determine possible clinical implications among this population.

Molecular diagnosis of tuberous sclerosis complex in fetuses and infants: an institutional case series.

OBJECTIVE: We describe the clinical and genetic characteristics of fetuses and infants diagnosed with tuberous sclerosis complex (TSC) in our centre, prenatally or neonatally, for a better understanding of the benefits of early screening. METHODS: In this retrospective study, we analysed the data on one fetus and nine infants with a definitive TSC diagnosis by genetic criteria (five patients carrying TSC1 variants and 5 patients carrying TSC2 variants). We explored the differences between phenotypes of patients carrying TSC1 and TSC2 pathogenic variants. RESULTS: The most common initial presenting features of TSC were cardiac rhabdomyomas (CRs) that were observed in nine out of ten patients. The most common postnatal features, besides CR, were presented with subependymal nodules-in five patients, and hypomelanotic macules-in four patients. In total, 10 variants causing TSC were detected in this study, including 5 novel variants. We demonstrated that patients with TSC2 variants had earlier onset and more severe clinical manifestations compared with patients carrying TSC1 variants. CONCLUSION: Early diagnosis of TSC improves genetic counselling and perinatal management.

Use of genome sequencing to hunt for cryptic second-hit variants: analysis of 31 cases recruited to the 100 000 Genomes Project.

BACKGROUND: Current clinical testing methods used to uncover the genetic basis of rare disease have inherent limitations, which can lead to causative pathogenic variants being missed. Within the rare disease arm of the 100 000 Genomes Project (100kGP), families were recruited under the clinical indication 'single autosomal recessive mutation in rare disease'. These participants presented with strong clinical suspicion for a specific autosomal recessive disorder, but only one suspected pathogenic variant had been identified through standard-of-care testing. Whole genome sequencing (WGS) aimed to identify cryptic 'second-hit' variants. METHODS: To investigate the 31 families with available data that remained unsolved following formal review within the 100kGP, SVRare was used to aggregate structural variants present in <1% of 100kGP participants. Small variants were assessed using population allele frequency data and SpliceAI. Literature searches and publicly available online tools were used for further annotation of pathogenicity. RESULTS: Using these strategies, 8/31 cases were solved, increasing the overall diagnostic yield of this cohort from 10/41 (24.4%) to 18/41 (43.9%). Exemplar cases include a patient with cystic fibrosis harbouring a novel exonic LINE1 insertion in CFTR and a patient with generalised arterial calcification of infancy with complex interlinked duplications involving exons 2-6 of ENPP1. Although ambiguous by short-read WGS, the ENPP1 variant structure was resolved using optical genome mapping and RNA analysis. CONCLUSION: Systematic examination of cryptic variants across a multi-disease cohort successfully identifies additional pathogenic variants. WGS data analysis in autosomal recessive rare disease should consider complex structural and small intronic variants as potentially pathogenic second hits.

Disease-specific therapy for the treatment of the cardiovascular manifestations of Fabry disease: a systematic review.

OBJECTIVE: The cardiovascular manifestations of Fabry disease are common and represent the leading cause of death. Disease-specific therapy, including enzyme replacement therapy (ERT) and chaperone therapy (migalastat), is recommended for patients exhibiting cardiovascular involvement, but its efficacy for modulating cardiovascular disease expression and optimal timing of initiation remains to be fully established. We therefore aimed to systematically review and evaluate the effectiveness of disease-specific therapy compared with placebo, and to no intervention, for the cardiovascular manifestations of Fabry disease. METHODS: Eight databases were searched from inception using a combination of relevant medical subject headings and keywords. Randomised, non-randomised studies with a comparator group and non-randomised studies without a comparator group were included. Studies were screened for eligibility and assessed for bias by two independent authors. The primary outcome comprised clinical cardiovascular events. Secondary outcomes included myocardial histology and measurements of cardiovascular structure, function and tissue characteristics. RESULTS: 72 studies were included, comprising 7 randomised studies of intervention, 16 non-randomised studies of intervention with a comparator group and 49 non-randomised studies of intervention without a comparator group. Randomised studies were not at serious risk of bias, but the others were at serious risk. Studies were highly heterogeneous in their design, outcome measurements and findings, which made assessment of disease-specific therapy effectiveness difficult. CONCLUSION: It remains unclear whether disease-specific therapy sufficiently impacts the cardiovascular manifestations of Fabry disease. Further work, ideally in larger cohorts, with more standardised clinical and phenotypic outcomes, the latter measured using contemporary techniques, are required to fully elucidate the cardiovascular impact of disease-specific therapy. PROSPERO REGISTRATION NUMBER: CRD42022295989.