Genetic Spectrum and Clinical Characteristics of Patients with Primary Ciliary Dyskinesia: a Belgian Single Center Study.

PURPOSE: We aimed to examine the correlation between clinical characteristics and the pathogenic gene variants in patients with Primary Ciliary Dyskinesia (PCD). METHODS: We conducted a retrospective single-center study in patients with PCD followed at the University Hospitals Leuven. We included patients with genetically confirmed PCD and described their genotype, data from ultrastructural ciliary evaluation and clinical characteristics. Genotype/phenotype correlations were studied in patients with the most frequently involved genes. RESULTS: We enrolled 74 patients with a median age of 25.58 years. The most frequently involved genes were DNAH11 (n = 23) and DNAH5 (n = 19). The most frequent types of pathogenic variants were missense (n = 42) and frameshift variants (n = 36) and most patients had compound heterozygous variants (n = 44). Ciliary ultrastructure (p < 0.001), situs (p = 0.015) and age at diagnosis (median 9.50 vs 4.71 years, p = 0.037) differed between DNAH11 and DNAH5. When correcting for situs this difference in age at diagnosis was no longer significant (p = 0.973). Patients with situs inversus were diagnosed earlier (p = 0.031). Respiratory tract microbiology (p = 0.161), lung function (cross-sectional, p = 0.829 and longitudinal, p = 0.329) and chest CT abnormalities (p = 0.202) were not significantly different between DNAH11 and DNAH5 variants. CONCLUSION: This study suggests a genotype-phenotype correlation for some of the evaluated clinical characteristics of the two most frequently involved genes in this study, namely DNAH11 and DNAH5.

Guidelines for Genetic Testing and Management of Alport Syndrome.

Genetic testing for pathogenic COL4A3-5 variants is usually undertaken to investigate the cause of persistent hematuria, especially with a family history of hematuria or kidney function impairment. Alport syndrome experts now advocate genetic testing for persistent hematuria, even when a heterozygous pathogenic COL4A3 or COL4A4 is suspected, and cascade testing of their first-degree family members because of their risk of impaired kidney function. The experts recommend too that COL4A3 or COL4A4 heterozygotes do not act as kidney donors. Testing for variants in the COL4A3-COL4A5 genes should also be performed for persistent proteinuria and steroid-resistant nephrotic syndrome due to suspected inherited FSGS and for familial IgA glomerulonephritis and kidney failure of unknown cause.

Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome: refining the ACMG criteria.

The recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3 and COL4A4 genes beyond the classical Alport phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3-5). It identified 'mutational hotspots' (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that 'well-established' functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3 and COL4A4 variants were common 'incidental' findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3-COL4A5 genes remains a challenge.

Next-generation sequencing in prenatal setting: Some examples of unexpected variant association.

The application of next-generation sequencing to fetal pathology has proved to increase the diagnostic yield in fetuses with abnormal ultrasounds. We retrospectively reviewed genetic data of 30 selected cases studied through targeted resequencing of OMIM genes. In our experience, clinical data proved to be essential to support diagnostic reasoning and enhance variants' assessment. The molecular diagnosis was reached in 19/30 (63%) cases. Only in 7/19 cases the molecular diagnosis confirmed the initial diagnostic hypothesis, showing the relevance of the genotype-first approach. According to the genotype-phenotype correlation, we were able to divide the solved cases into three groups: i) the correlation is well established but it was missed due to lack of specificity, unusual presentation or recent description; ii) the clinical presentation is much more severe than currently known for the underlying condition; iii) the correlation does not recapitulate the entire phenotype, possibly due to the fetal presentation or multiple coexisting conditions. Moreover, we found a higher proportion of recessive diagnosis in abnormal fetuses compared to cohorts of individuals with developmental delay. Our findings suggest that fetal pathology may be enriched in rare alleles and/or in unusual combinations, counter-selected in postnatal genomes and thus contributing to both phenotypic extremeness and atypical presentation.

BCAP31-related syndrome: The first de novo report.

Pathogenic variants in the BCAP31 gene have recently been associated with a severe congenital neurological phenotype, named DDCH after its key features: deafness, dystonia and central hypomyelination. BCAP31 is located at the Xq28 chromosomal region and only male individuals are currently known to be affected, the pathogenic variant being usually transmitted by healthy mothers. Here, we describe a three-year-old male child referred for severe developmental delay, failure to thrive, hearing loss and dyskinetic movements. After a conventional diagnostic workflow, including a normal array-CGH, a tentative diagnosis of dyskinetic cerebral palsy was retained. Clinical exome sequencing in the trio identified a small intragenic deletion in exon 8 of BCAP31, c.709_721del (p.Val237Trpfs*69), originated de novo and not previously reported. Based on the ACMG variant classification, this variant is predicted to be 'likely pathogenic'. Given the consistent phenotypical overlap with the subjects already ascertained with DDCH, we considered this variant to be clinically relevant for this child and causative of his condition.

Heterozygous loss-of-function variants of MEIS2 cause a triad of palatal defects, congenital heart defects, and intellectual disability.

Deletions on chromosome 15q14 are a known chromosomal cause of cleft palate, typically co-occurring with intellectual disability, facial dysmorphism, and congenital heart defects. The identification of patients with loss-of-function variants in MEIS2, a gene within this deletion, suggests that these features are attributed to haploinsufficiency of MEIS2. To further delineate the phenotypic spectrum of the MEIS2-related syndrome, we collected 23 previously unreported patients with either a de novo sequence variant in MEIS2 (9 patients), or a 15q14 microdeletion affecting MEIS2 (14 patients). All but one de novo MEIS2 variant were identified by whole-exome sequencing. One variant was found by targeted sequencing of MEIS2 in a girl with a clinical suspicion of this syndrome. In addition to the triad of palatal defects, heart defects, and developmental delay, heterozygous loss of MEIS2 results in recurrent facial features, including thin and arched eyebrows, short alae nasi, and thin vermillion. Genotype-phenotype comparison between patients with 15q14 deletions and patients with sequence variants or intragenic deletions within MEIS2, showed a higher prevalence of moderate-to-severe intellectual disability in the former group, advocating for an independent locus for psychomotor development neighboring MEIS2.

Massive parallel sequencing identifies RAPSN and PDHA1 mutations causing fetal akinesia deformation sequence.

INTRODUCTION: Fetal akinesia deformation sequence (FADS) or arthrogryposis multiplex congenita (AMC) is characterized by clinical ambiguity and genetic heterogeneity, hampering genetic diagnosis via traditional sequencing methods. Next generation sequencing (NGS) of all known disease-causing genes offers an elegant solution to identify the genetic etiology of AMC/FADS in a diagnostic setting. METHODS: An in-house developed disease-associated gene panel was conducted in two unrelated fetuses with FADS. First, a de novo analysis was performed on the entire disease-associated gene panel. If no pathogenic mutation was identified, analysis of variants retained in a specific subpanel with arthrogryposis/fetal akinesia-causing genes was performed. RESULTS: In the first family, FADS relates to a homozygous c.484G > A (p.Glu162Lys) mutation in the gene RAPSN. The second case concerns a sporadic patient with brain anomalies and arthrogryposis due to a de novo hemizygous c.498C > T splice-site mutation in the pyruvate dehydrogenase-alpha 1 (PDHA1) gene. DISCUSSION: NGS facilitated genetic diagnosis, and hence genetic counseling, for both families with AMC/FADS. Biallelic RAPSN mutations typically result in congenital myasthenia syndrome, or occasionally in FADS. This is the first report attributing the RAPSN mutation c.484G > A, identified in a homozygous state in patient 1, to FADS. The second patient represents the first case of AMC due to a PDHA1 mutation, advocating that pyruvate dehydrogenase deficiency should be considered in the differential diagnosis of fetal akinesia. This study illustrates the relevance of a disease-associated-gene panel as a diagnostic tool in pregnancies complicated by this genetically heterogeneous condition.