Severe congenital contractural arachnodactyly caused by biallelic pathogenic variants in FBN2.

Fibrillin-2, encoded by FBN2, plays an important role in the early process of elastic fiber assembly. To date, heterozygous pathogenic variants in FBN2 have been shown to cause congenital contractural arachnodactyly (CCA; Beals-Hecht syndrome). Classical CCA is characterized by long and slender fingers and toes, ear deformities, joint contractures at birth, clubfeet, muscular hypoplasia and often tall stature. In individuals with a severe CCA form, different cardiovascular or gastrointestinal anomalies have been described. Here, we report on a 15-year-old girl with a severe form of CCA and novel biallelic variants in FBN2. The girl inherited the missense variant c.3563G > T/p.(Gly1188Val) from her unaffected father and the nonsense variant c.6831C > A/p.(Cys2277*) from her healthy mother. We could detect only a small amount of FBN2 transcripts harboring the nonsense variant in leukocyte-derived mRNA from the patient and mother suggesting nonsense-mediated mRNA decay. As the father did not show any clinical signs of CCA we hypothesize the missense variant c.3563G > T to be a hypomorphic allele. Taken together, our data suggests that severe CCA can be inherited in an autosomal-recessive manner by compound heterozygosity of a hypomorphic and a null allele of the FBN2 gene.

Clinically relevant variants in a large cohort of Indian patients with Marfan syndrome and related disorders identified by next-generation sequencing.

Marfan syndrome and related disorders are a group of heritable connective tissue disorders and share many clinical features that involve cardiovascular, skeletal, craniofacial, ocular, and cutaneous abnormalities. The majority of affected individuals have aortopathies associated with early mortality and morbidity. Implementation of targeted gene panel next-generation sequencing in these individuals is a powerful tool to obtain a genetic diagnosis. Here, we report on clinical and genetic spectrum of 53 families from India with a total of 83 patients who had a clinical diagnosis suggestive of Marfan syndrome or related disorders. We obtained a molecular diagnosis in 45/53 (85%) index patients, in which 36/53 (68%) had rare variants in FBN1 (Marfan syndrome; 63 patients in total), seven (13.3%) in TGFBR1/TGFBR2 (Loeys-Dietz syndrome; nine patients in total) and two patients (3.7%) in SKI (Shprintzen-Goldberg syndrome). 21 of 41 rare variants (51.2%) were novel. We did not detect a disease-associated variant in 8 (15%) index patients, and none of them met the Ghent Marfan diagnostic criteria. We found the homozygous FBN1 variant p.(Arg954His) in a boy with typical features of Marfan syndrome. Our study is the first reporting on the spectrum of variants in FBN1, TGFBR1, TGFBR2, and SKI in Indian individuals.

Next-generation sequencing of 32 genes associated with hereditary aortopathies and related disorders of connective tissue in a cohort of 199 patients.

PURPOSE: Heritable factors play an important etiologic role in connective tissue disorders (CTD) with vascular involvement, and a genetic diagnosis is getting increasingly important for gene-tailored, personalized patient management. METHODS: We analyzed 32 disease-associated genes by using targeted next-generation sequencing and exome sequencing in a clinically relevant cohort of 199 individuals. We classified and refined sequence variants according to their likelihood for pathogenicity. RESULTS: We identified 1 pathogenic variant (PV; in FBN1 or SMAD3) in 15 patients (7.5%) and ≥1 likely pathogenic variant (LPV; in COL3A1, FBN1, FBN2, LOX, MYH11, SMAD3, TGFBR1, or TGFBR2) in 19 individuals (9.6%), together resulting in 17.1% diagnostic yield. Thirteen PV/LPV were novel. Of PV/LPV-negative patients 47 (23.6%) showed ≥1 variant of uncertain significance (VUS). Twenty-five patients had concomitant variants. In-depth evaluation of reported/calculated variant classes resulted in reclassification of 19.8% of variants. CONCLUSION: Variant classification and refinement are essential for shaping mutational spectra of disease genes, thereby improving clinical sensitivity. Obligate stringent multigene analysis is a powerful tool for identifying genetic causes of clinically related CTDs. Nonetheless, the relatively high rate of PV/LPV/VUS-negative patients underscores the existence of yet unknown disease loci and/or oligogenic/polygenic inheritance.

Clinical spectrum of females with HCCS mutation: from no clinical signs to a neonatal lethal form of the microphthalmia with linear skin defects (MLS) syndrome.

BACKGROUND: Segmental Xp22.2 monosomy or a heterozygous HCCS mutation is associated with the microphthalmia with linear skin defects (MLS) or MIDAS (microphthalmia, dermal aplasia, and sclerocornea) syndrome, an X-linked disorder with male lethality. HCCS encodes the holocytochrome c-type synthase involved in mitochondrial oxidative phosphorylation (OXPHOS) and programmed cell death. METHODS: We characterized the X-chromosomal abnormality encompassing HCCS or an intragenic mutation in this gene in six new female patients with an MLS phenotype by cytogenetic analysis, fluorescence in situ hybridization, sequencing, and quantitative real-time PCR. The X chromosome inactivation (XCI) pattern was determined and clinical data of the patients were reviewed. RESULTS: Two terminal Xp deletions of ≥ 11.2 Mb, two submicroscopic copy number losses, one of ~850 kb and one of ≥ 3 Mb, all covering HCCS, 1 nonsense, and one mosaic 2-bp deletion in HCCS are reported. All females had a completely (>98:2) or slightly skewed (82:18) XCI pattern. The most consistent clinical features were microphthalmia/anophthalmia and sclerocornea/corneal opacity in all patients and congenital linear skin defects in 4/6. Additional manifestations included various ocular anomalies, cardiac defects, brain imaging abnormalities, microcephaly, postnatal growth retardation, and facial dysmorphism. However, no obvious clinical sign was observed in three female carriers who were relatives of one patient. CONCLUSION: Our findings showed a wide phenotypic spectrum ranging from asymptomatic females with an HCCS mutation to patients with a neonatal lethal MLS form. Somatic mosaicism and the different ability of embryonic cells to cope with an OXPHOS defect and/or enhanced cell death upon HCCS deficiency likely underlie the great variability in phenotypes.

Mucopolysaccharidosis type II in a female carrying a heterozygous stop mutation of the iduronate-2-sulfatase gene and showing a skewed X chromosome inactivation.

We report a Mexican girl showing the full blown clinical picture of mucopolysaccharidosis type II (MPSII). Iduronate-2-sulfatase (IDS) activity was low and she carried a heterozygous de novo c.1327C>T transition in exon 9, that changes codon 443 for a premature stop (TGA; p.Arg443(*)). Analysis of X-chromosome inactivation in androgen receptor (AR) locus showed a highly skewed ratio of 92:8 suggesting a functional hemizygosity with dominant expression of the mutant IDS and explaining the disease manifestation. This is one of the rare cases of females affected by MPSII due to the combined effect of a skewed X-chromosome inactivation and a de novo IDS mutation. We recommend that clinicians should consider the diagnosis of MPSII even in a girl without positive family history for this condition.

Phenotypic spectrum associated with CASK loss-of-function mutations.

BACKGROUND: Heterozygous mutations in the CASK gene in Xp11.4 have been shown to be associated with a distinct brain malformation phenotype in females, including disproportionate pontine and cerebellar hypoplasia. METHODS: The study characterised the CASK alteration in 20 new female patients by molecular karyotyping, fluorescence in situ hybridisation, sequencing, reverse transcriptase (RT) and/or quantitative real-time PCR. Clinical and brain imaging data of a total of 25 patients were reviewed. RESULTS: 11 submicroscopic copy number alterations, including nine deletions of ~11 kb to 4.5 Mb and two duplications, all covering (part of) CASK, four splice, four nonsense, and one 1 bp deletion are reported. These heterozygous CASK mutations most likely lead to a null allele. Brain imaging consistently showed diffuse brainstem and cerebellar hypoplasia with a dilated fourth ventricle, but of remarkably varying degrees. Analysis of 20 patients in this study, and five previously reported patients, revealed a core clinical phenotype comprising severe developmental delay/intellectual disability, severe postnatal microcephaly, often associated with growth retardation, (axial) hypotonia with or without hypertonia of extremities, optic nerve hypoplasia, and/or other eye abnormalities. A recognisable facial phenotype emerged, including prominent and broad nasal bridge and tip, small or short nose, long philtrum, small chin, and/or large ears. CONCLUSIONS: These findings define the phenotypic spectrum associated with CASK loss-of-function mutations. The combination of developmental and brain imaging features together with mild facial dysmorphism is highly suggestive of this disorder and should prompt subsequent testing of the CASK gene.

Nonsense mutations in SMPX, encoding a protein responsive to physical force, result in X-chromosomal hearing loss.

The fact that hereditary hearing loss is the most common sensory disorder in humans is reflected by, among other things, an extraordinary allelic and nonallelic genetic heterogeneity. X-chromosomal hearing impairment represents only a minor fraction of all cases. In a study of a Spanish family the locus for one of the X-chromosomal forms was assigned to Xp22 (DFNX4). We mapped the disease locus in the same chromosomal region in a large German pedigree with X-chromosomal nonsyndromic hearing impairment by using genome-wide linkage analysis. Males presented with postlingual hearing loss and onset at ages 3-7, whereas onset in female carriers was in the second to third decades. Targeted DNA capture with high-throughput sequencing detected a nonsense mutation in the small muscle protein, X-linked (SMPX) of affected individuals. We identified another nonsense mutation in SMPX in patients from the Spanish family who were previously analyzed to map DFNX4. SMPX encodes an 88 amino acid, cytoskeleton-associated protein that is responsive to mechanical stress. The presence of Smpx in hair cells and supporting cells of the murine cochlea indicates its role in the inner ear. The nonsense mutations detected in the two families suggest a loss-of-function mechanism underlying this form of hearing impairment. Results obtained after heterologous overexpression of SMPX proteins were compatible with this assumption. Because responsivity to physical force is a characteristic feature of the protein, we propose that long-term maintenance of mechanically stressed inner-ear cells critically depends on SMPX function.

Autosomal-recessive posterior microphthalmos is caused by mutations in PRSS56, a gene encoding a trypsin-like serine protease.

Posterior microphthalmos (MCOP) is a rare isolated developmental anomaly of the eye characterized by extreme hyperopia due to short axial length. The population of the Faroe Islands shows a high prevalence of an autosomal-recessive form (arMCOP) of the disease. Based on published linkage data, we refined the position of the disease locus (MCOP6) in an interval of 250 kb in chromosome 2q37.1 in two large Faroese families. We detected three different mutations in PRSS56. Patients of the Faroese families were either homozygous for c.926G>C (p.Trp309Ser) or compound heterozygous for c.926G>C and c.526C>G (p.Arg176Gly), whereas a homozygous 1 bp duplication (c.1066dupC) was identified in five patients with arMCOP from a consanguineous Tunisian family. In one patient with MCOP from the Faroe Islands and in another one from Turkey, no PRSS56 mutation was detected, suggesting nonallelic heterogeneity of the trait. Using RT-PCR, PRSS56 transcripts were detected in samples derived from the human adult retina, cornea, sclera, and optic nerve. The expression of the mouse ortholog could be first detected in the eye at E17 and was maintained into adulthood. The predicted PRSS56 protein is a 603 amino acid long secreted trypsin-like serine peptidase. The c.1066dupC is likely to result in a functional null allele, whereas the two point mutations predict the replacement of evolutionary conserved and functionally important residues. Molecular modeling of the p.Trp309Ser mutant suggests that both the affinity and reactivity of the enzyme toward in vivo protein substrates are likely to be substantially reduced.

Nonsense mutations in FAM161A cause RP28-associated recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is a degenerative disease of the retina leading to progressive loss of vision and, in many instances, to legal blindness at the end stage. The RP28 locus was assigned in 1999 to the short arm of chromosome 2 by homozygosity mapping in a large Indian family segregating autosomal-recessive RP (arRP). Following a combined approach of chromatin immunoprecipitation and parallel sequencing of genomic DNA, we identified a gene, FAM161A, which was shown to carry a homozygous nonsense mutation (p.Arg229X) in patients from the original RP28 pedigree. Another homozygous FAM161A stop mutation (p.Arg437X) was detected in three subjects from a cohort of 118 apparently unrelated German RP patients. Age at disease onset in these patients was in the second to third decade, with severe visual handicap in the fifth decade and legal blindness in the sixth to seventh decades. FAM161A is a phylogenetically conserved gene, expressed in the retina at relatively high levels and encoding a putative 76 kDa protein of unknown function. In the mouse retina, Fam161a mRNA is developmentally regulated and controlled by the transcription factor Crx, as demonstrated by chromatin immunoprecipitation and organotypic reporter assays on explanted retinas. Fam161a protein localizes to photoreceptor cells during development, and in adult animals it is present in the inner segment as well as the outer plexiform layer of the retina, the synaptic interface between photoreceptors and their efferent neurons. Taken together, our data indicate that null mutations in FAM161A are responsible for the RP28-associated arRP.