Functional,structural, and genetic evaluation of 20 CDKN2A germ line mutations identified in melanoma‐prone families or patients

Germline mutations of the CDKN2A gene are found in melanoma‐prone families and individuals with multiple sporadic melanomas. The encoded protein, p16^INK4A, comprises four ankyrin‐type repeats, and the mutations, most of which are missense and occur throughout the entire coding region, can disrupt the conformation of these structural motifs as well as the association of p16^INK4a with its physiological targets, the cyclin‐dependent kinases (CDKs) CDK4 and CDK6. Assessing pathogenicity of nonsynonymous mutations is critical to evaluate melanoma risk in carriers. In the current study, we investigate 20 CDKN2A germline mutations whose effects on p16^INK4A structure and function have not been previously documented (Thr18_Ala19dup, Gly23Asp, Arg24Gln, Gly35Ala, Gly35Val, Ala57Val, Ala60Val, Ala60Arg, Leu65dup, Gly67Arg, Gly67_Asn71del, Glu69Gly, Asp74Tyr, Thr77Pro, Arg80Pro, Pro81Thr, Arg87Trp, Leu97Arg, Arg99Pro, and [Leu113Leu;Pro114Ser]). By considering genetic information, the predicted impact of each variant on the protein structure, its ability to interact with CDK4 and impede cell proliferation in experimental settings, we conclude that 18 of the 20 CDKN2A variants can be classed as loss of function mutations, whereas the results for two remain ambiguous. Discriminating between mutant and neutral variants of p16^INK4A not only adds to our understanding of the functionally critical residues in the protein but provides information that can be used for melanoma risk prediction. Hum Mutat 0, 1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Novel phenotype of achondroplasia due to biallelic FGFR3 pathogenic variants

Pathogenic variants in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for a broad spectrum of skeletal dysplasias, including achondroplasia (ACH). The classic phenotype of ACH is caused by two highly prevalent mutations, c.1138G > A and c.1138G > C (p.Gly380Arg). In the homozygous state, these variant results in a severe skeletal dysplasia, neurologic deficits, and early demise from respiratory insufficiency. Although homozygous biallelic mutations have been reported in patients with ACH in combination with hypochondroplasia or other dominant skeletal dysplasias, thus far, no cases of heterozygous biallelic pathogenic ACH‐related variants in FGFR3 have been reported. We describe a novel phenotype of an infant with two ACH‐related mutations in FGFR3, p.Gly380Arg and p.Ser344Cys. Discordant features from classic ACH include atypical radiographic findings, severe obstructive sleep apnea, and focal, migrating seizures. We also report the long‐term clinical course of her father, who harbors the p.Ser344Cys mutation that has only been reported once previously in a Japanese patient. The phenotype of heterozygous biallelic mutations in FGFR3 associated with ACH is variable, underscoring the importance of recognition and accurate diagnosis to ensure appropriate management.     

Developmental and epileptic encephalopathy in two siblings with a novel,homozygous missense variant in SCN1B

Developmental and epileptic encephalopathies are genetic disorders in which both the developmental disability and the frequent epileptic activity are the effect of a specific gene variant. While heterozygous variants in SCN1B have been described in families with generalized epilepsy with febrile seizures plus, Type 1, only three cases of homozygous, missense variants in SCN1B have been reported in association with autosomal recessive inheritance of a severe developmental and epileptic encephalopathy. We present two siblings who are homozygous for a novel, missense variant in SCN1B, c.265C>T, predicting p.Arg89Cys. The proband is an 11‐year‐old female with infantile‐onset, fever‐induced, intractable generalized tonic–clonic seizures, myoclonic seizures, and developmental slowing and autism spectrum disorder occurring later in the course of the disease. Her 4‐year‐old brother had a similar epilepsy phenotype, but still displays normal development. This variant has not been previously reported in the homozygous state in control databases. The variant was predicted to be damaging and occurred in the vicinity of other epileptic encephalopathy‐associated missense variants that are biallelic and located in the extracellular immunoglobulin loop domain of the protein, which mediates interaction of the beta‐1 subunit with cellular adhesion molecules. Our report is the first set of siblings with homozygosity for the p.Arg89Cys variant in SCN1B and further implicates biallelic mutations in this gene as a cause of epileptic encephalopathy mimicking Dravet syndrome. Interestingly, the phenotype we observed was milder compared to that previously described in patients with recessive SCN1B mutations.     

IL11RA‐related Crouzon‐like autosomal recessive craniosynostosis in 10 new patients: Resemblances and differences

By describing 10 new patients recruited in centres for Human Genetics, we further delineate the clinical spectrum of a Crouzon‐like craniosynostosis disorder, officially termed craniosynostosis and dental anomalies (MIM614188). Singularly, it is inherited according to an autosomal recessive mode of inheritance. We identified six missense mutations in IL11RA, a gene encoding the alpha subunit of interleukin 11 receptor, 4 of them being novel, including 2 in the Ig‐like C2‐type domain. A subset of patients had an associated connective tissue disorder with joint hypermobility and intervertebral discs fragility. A smaller number of teeth anomalies than that previously reported in the two large series of patients evaluated in dental institutes points toward an ascertainment bias.     

A genetic and clinical study of individuals with nonsyndromic retinopathy consequent upon sequence variants in HGSNAT,the gene associated with Sanfilippo C mucopolysaccharidosis

Pathogenic variants in the gene HGSNAT (heparan‐α‐glucosaminide N‐acetyltransferase) have been reported to underlie two distinct recessive conditions, depending on the specific genotype, mucopolysaccharidosis type IIIC (MPSIIIC)—a severe childhood‐onset lysosomal storage disorder, and adult‐onset nonsyndromic retinitis pigmentosa (RP). Here we describe the largest cohort to‐date of HGSNAT‐associated nonsyndromic RP patients, and describe their retinal phenotype, leukocyte enzymatic activity, and likely pathogenic genotypes. We identified biallelic HGSNAT variants in 17 individuals (15 families) as the likely cause of their RP. None showed any other symptoms of MPSIIIC. All had a mild but significant reduction of HGSNAT enzyme activity in leukocytes. The retinal condition was generally of late‐onset, showing progressive degeneration of a concentric area of paramacular retina, with preservation but reduced electroretinogram responses. Symptoms, electrophysiology, and imaging suggest the rod photoreceptor to be the cell initially compromised. HGSNAT enzymatic testing was useful in resolving diagnostic dilemmas in compatible patients. We identified seven novel sequence variants [p.(Arg239Cys); p.(Ser296Leu); p.(Phe428Cys); p.(Gly248Ala); p.(Gly418Arg), c.1543‐2A>C; c.1708delA], three of which were considered to be retina‐disease‐specific alleles. The most prevalent retina‐disease‐specific allele p.(Ala615Thr) was observed heterozygously or homozygously in 8 and 5 individuals respectively (7 and 4 families). Two siblings in one family, while identical for the HGSNAT locus, but discordant for retinal disease, suggest the influence of trans‐acting genetic or environmental modifying factors.     

Expanding the clinical spectrum of biallelic ZNF335 variants

ZNF335 plays an essential role in neurogenesis and biallelic variants in ZNF335 have been identified as the cause of severe primary autosomal recessive microcephaly in 2 unrelated families. We describe, herein, 2 additional affected individuals with biallelic ZNF335 variants, 1 individual with a homozygous c.1399 T > C, p.(Cys467Arg) variant, and a second individual with compound heterozygous c.2171_2173delTCT, p.(Phe724del) and c.3998A > G, p.(Glu1333Gly) variants with the latter variant predicted to affect splicing. Whereas the first case presented with early death and a severe phenotype characterized by anterior agyria with prominent extra‐axial spaces, absent basal ganglia, and hypoplasia of the brainstem and cerebellum, the second case had a milder clinical presentation with hypomyelination and otherwise preserved brain structures on MRI. Our findings expand the clinical spectrum of ZNF335‐associated microcephaly.     

Siblings with profound connective tissue disease: First report of biallelic TGFBR1-related Loeys-Dietz syndrome

Heterozygous missense variants in TGFBR1, encoding one subunit of the transforming growth factor-beta receptor, are a well-established cause of Loeys-Dietz syndrome (LDS)—an autosomal dominant disorder with variable phenotypic expression. Patients with LDS have compromised connective tissues that can result in life-threatening arterial aneurysms, craniosynostosis, characteristic craniofacial and skeletal anomalies, skin translucency, and abnormal wound healing. We report a full sibship with a biallelic type of TGFBR1-related disease. Each born at 38 weeks had aortic root dilation, congenital diaphragmatic hernia (CDH), skin translucency, and profound joint laxity at birth. Both had progressive dilation of the aorta and recurrence of a diaphragmatic defect after plication early in infancy. Patient 1 died at 66 days of age and Patient 2 is alive at 4 years and 4 months of age with multiple morbidities including cystic lung disease complicated by recurrent pneumothoraces and ventilator dependence, craniosynostosis, cervical spine instability, progressive dilation of the aorta, worsening pectus excavatum, large lateral abdominal wall hernia, and diffuse aortic ectasia. Fibroblasts cultured from Patient 2 showed decreased TGF-β responsiveness when compared to control fibroblasts, consistent with previous observations in cells from individuals with autosomal dominant LDS. Whole genome copy number evaluation and sequencing for both patients including their parents as reference revealed compound heterozygous variants of uncertain clinical significance in exon 2 of TGFBR1 (c.239G>A; p.Arg80Gln paternal and c.313C>G; p.His105Asp maternal) in both siblings in trans. Each parent with their respective variant has no apparent medical issues and specifically no LDS characteristics. Neither of these variants have been previously reported. Thousands of patients have been diagnosed with LDS—an established autosomal dominant disease. These siblings represent the first reports of biallelic TGFBR1-related LDS and expand the differential diagnosis of CDH.     

Duplications in the G3 domain or switch II region in HRAS identified in patients with Costello syndrome

Costello syndrome (CS) is an autosomal-dominant disorder characterized by distinctive facial features, hypertrophic cardiomyopathy, skeletal abnormalities, intellectual disability, and predisposition to cancers. Germline variants in HRAS have been identified in patients with CS. Intragenic HRAS duplications have been reported in three patients with a milder phenotype of CS. In this study, we identified two known HRAS variants, p.(Glu63_Asp69dup), p.(Glu62_Arg68dup), and one novel HRAS variant, p.(Ile55_Asp57dup), in patients with CS, including a patient with craniosynostosis. These intragenic duplications are located in the G3 domain and the switch II region. Cells expressing cDNA with these three intragenic duplications showed an increase in ELK-1 transactivation. Injection of wild-type or mutant HRAS mRNAs with intragenic duplications in zebrafish embryos showed significant elongation of the yolk at 11 h postfertilization, which was improved by MEK inhibitor treatment, and a variety of developmental abnormalities at 3 days post fertilization was observed. These results indicate that small in-frame duplications affecting the G3 domain and switch II region of HRAS increase the activation of the ERK pathway, resulting in developmental abnormalities in zebrafish or patients with CS.     

Biallelic and De Novo Variants in DONSON Reveal a Clinical Spectrum of Cell Cycle‐opathies with Microcephaly,Dwarfism and Skeletal Abnormalities

Co‐occurrence of primordial dwarfism and microcephaly together with particular skeletal findings are seen in a wide range of Mendelian syndromes including microcephaly micromelia syndrome (MMS, OMIM 251230), microcephaly, short stature, and limb abnormalities (MISSLA, OMIM 617604), and microcephalic primordial dwarfisms (MPDs). Genes associated with these syndromes encode proteins that have crucial roles in DNA replication or in other critical steps of the cell cycle that link DNA replication to cell division. We identified four unrelated families with five affected individuals having biallelic or de novo variants in DONSON presenting with a core phenotype of severe short stature (z score < ?3 SD), additional skeletal abnormalities, and microcephaly. Two apparently unrelated families with identical homozygous c.631C > T p.(Arg211Cys) variant had clinical features typical of Meier‐Gorlin syndrome (MGS), while two siblings with compound heterozygous c.346delG p.(Asp116Ile*62) and c.1349A > G p.(Lys450Arg) variants presented with Seckel‐like phenotype. We also identified a de novo c.683G > T p.(Trp228Leu) variant in DONSON in a patient with prominent micrognathia, short stature and hypoplastic femur and tibia, clinically diagnosed with Femoral‐Facial syndrome (FFS, OMIM 134780). Biallelic variants in DONSON have been recently described in individuals with microcephalic dwarfism. These studies also demonstrated that DONSON has an essential conserved role in the cell cycle. Here we describe novel biallelic and de novo variants that are associated with MGS, Seckel‐like phenotype and FFS, the last of which has not been associated with any disease gene to date.     

FOXE3 mutations: genotype‐phenotype correlations

Microphthalmia and anophthalmia (MA) are severe developmental eye anomalies, many of which are likely to have an underlying genetic cause. More than 30 genes have been described, each of which is responsible for a small percentage of these anomalies. Among these, is the FOXE3 gene, which was initially described in individuals with dominantly inherited anterior segment dysgenesis and, subsequently, associated with recessively inherited primary aphakia, sclerocornea and microphthalmia. In this work, we describe 8 individuals presenting with an MA phenotype. Among them, 7 are carrying biallelic recessive FOXE3 mutations and 2 of these have novel mutations: p.(Ala78Thr) and p.(Arg104Cys). The last of our patients is carrying in the heterozygous state the recessive p.(Arg90Leu) mutation in the FOXE3 gene. To further understand FOXE3 involvement in this wide spectrum of ocular anomalies with 2 different patterns of inheritance, we reviewed all individuals with ocular abnormalities described in the literature for which a FOXE3 mutation was identified. This review demonstrates that correlations exist between the mutation type, mode of inheritance and the phenotype severity. Furthermore, understanding the genetic basis of these conditions will contribute to overall understanding of eye development, improve the quality of care, genetic counseling and, in future, gene‐based therapies.     

Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A)

Defects in the motor domain of kinesin family member 1A (KIF1A), a neuron‐specific ATP‐dependent anterograde axonal transporter of synaptic cargo, are well‐recognized to cause a spectrum of neurological conditions, commonly known as KIF1A‐associated neurological disorders (KAND). Here, we report one mutation‐negative female with classic Rett syndrome (RTT) harboring a de novo heterozygous novel variant [NP_001230937.1:p.(Asp248Glu)] in the highly conserved motor domain of KIF1A. In addition, three individuals with severe neurodevelopmental disorder along with clinical features overlapping with KAND are also reported carrying de novo heterozygous novel [NP_001230937.1:p.(Cys92Arg) and p.(Pro305Leu)] or previously reported [NP_001230937.1:p.(Thr99Met)] variants in KIF1A. In silico tools predicted these variants to be likely pathogenic, and 3D molecular modeling predicted defective ATP hydrolysis and/or microtubule binding. Using the neurite tip accumulation assay, we demonstrated that all novel KIF1A variants significantly reduced the ability of the motor domain of KIF1A to accumulate along the neurite lengths of differentiated SH‐SY5Y cells. In vitro microtubule gliding assays showed significantly reduced velocities for the variant p.(Asp248Glu) and reduced microtubule binding for the p.(Cys92Arg) and p.(Pro305Leu) variants, suggesting a decreased ability of KIF1A to move along microtubules. Thus, this study further expanded the phenotypic characteristics of KAND individuals with pathogenic variants in the KIF1A motor domain to include clinical features commonly seen in RTT individuals.     

Functional profiling of LDLR variants: Important evidence for variant classification: Functional profiling of LDLR variants

BackgroundFamilial Hypercholesterolemia (FH) is a semidominant disorder of the lipid metabolism associated with premature atherosclerosis and coronary heart disease. So far, about 3,000 unique LDLR variants have been described, most of which lack functional evidence proving their effect on LDLR function, despite the important role that functional studies play in variant classification.ObjectiveIn this work, we aimed to functionally characterize 13 rare missense variants, identified worldwide and in Portugal, in clinical FH patients.MethodsLDLR-deficient CHO-ldlA7 cells were transfected with plasmids carrying different LDLR variants generated by site-directed mutagenesis. LDLR activity and expression were assessed by FACS.Results11/13 variants affect LDLR function (p.Cys109Phe; p.Cys143Arg; p.Glu267Lys; p.Cys352Ser; p.Ile451Thr; p.His485Gln; p.Asp492Asn; p.Val500Ala; p.Gly529Arg; p.Phe614Ile; p.Glu626Lys) and 2/13 are inconclusive (p.Arg81Cys; p.Gly98Arg;).ConclusionOf the 13 variants studied, 8 were classified as VUS by ACMG criteria, but for 7 of these 8, our functional studies were able to reassign them as Likely pathogenic or Pathogenic. For an accurate diagnosis, an effort must be made to improve functional characterization of putative disease-causing variants.     

The spectrum of BRCA1 and BRCA2 pathogenic sequence variants in Middle Eastern,North African,and South European countries

BRCA1 BRCA2 mutational spectrum in the Middle East, North Africa, and Southern Europe is not well characterized. The unique history and cultural practices characterizing these regions, often involving consanguinity and inbreeding, plausibly led to the accumulation of population‐specific founder pathogenic sequence variants (PSVs). To determine recurring BRCA PSVs in these locales, a search in PUBMED, EMBASE, BIC, and CIMBA was carried out combined with outreach to researchers from the relevant countries for unpublished data. We identified 232 PSVs in BRCA1 and 239 in BRCA2 in 25 of 33 countries surveyed. Common PSVs that were detected in four or more countries were c.5266dup (p.Gln1756Profs), c.181T>G (p.Cys61Gly), c.68_69del (p.Glu23Valfs), c.5030_5033del (p.Thr1677Ilefs), c.4327C>T (p.Arg1443Ter), c.5251C>T (p.Arg1751Ter), c.1016dup (p.Val340Glyfs), c.3700_3704del (p.Val1234Glnfs), c.4065_4068del (p.Asn1355Lysfs), c.1504_1508del (p.Leu502Alafs), c.843_846del (p.Ser282Tyrfs), c.798_799del (p.Ser267Lysfs), and c.3607C>T (p.Arg1203Ter) in BRCA1 and c.2808_2811del (p.Ala938Profs), c.5722_5723del (p.Leu1908Argfs), c.9097dup (p.Thr3033Asnfs), c.1310_1313del (p. p.Lys437Ilefs), and c.5946del (p.Ser1982Argfs) for BRCA2. Notably, some mutations (e.g., p.Asn257Lysfs (c.771_775del)) were observed in unrelated populations. Thus, seemingly genotyping recurring BRCA PSVs in specific populations may provide first pass BRCA genotyping platform.     

Biallelic variants p.Arg1133Cys and p.Arg1379Cys in COL2A1: Further delineation of phenotypic spectrum of recessive Type 2 collagenopathies

The phenotypic spectrum of Type 2 collagenopathies ranges from lethal achondrogenesis Type 2 to milder osteoarthritis with mild chondrodysplasia. All of them are monoallelic except for the two recent reports showing that biallelic variants in COL2A1 can cause spondyloepiphyseal dysplasia congenita in two children. Here we report two additional families with homozygous variants, c.4135C>T (p.Arg1379Cys) and c.3190C>T (p.Arg1133Cys) in COL2A1 resulting in two distinct skeletal dysplasia phenotypes of intermediate severity. Though all six patients from four families exhibit a spondylo‐epimetaphyseal dysplasia, they demonstrate a wide variation in severity of short stature and involvement of epiphyses, metaphyses, and vertebrae. We hypothesize that the variants are likely to be hypomorphic, given the underlying mechanisms of disease causation for known heterozygous variants in COL2A1. With this report, we provide further evidence to the existence of autosomal recessive Type 2 collagenopathy.     

The expanding clinical phenotype of germline ABL1‐associated congenital heart defects and skeletal malformations syndrome

Congenital heart defects and skeletal malformations syndrome (CHDSKM) is a rare autosomal dominant disorder characterized by congenital heart disease, skeletal abnormalities, and failure to thrive. CHDSKM is caused by germline mutations in ABL1. To date, three variants have been in association with CHDSKM. In this study, we describe three de novo missense variants, c.407C>T (p.Thr136Met), c.746C>T (p.Pro249Leu), and c.1573G>A (p.Val525Met), and one recurrent variant, c.1066G>A (p.Ala356Thr), in six patients, thereby expanding the phenotypic spectrum of CHDSKM to include hearing impairment, lipodystrophy‐like features, renal hypoplasia, and distinct ocular abnormalities. Functional investigation of the three novel variants showed an increased ABL1 kinase activity. The cardiac findings in additional patients with p.Ala356Thr contribute to the accumulating evidence that patients carrying either one of the recurrent variants, p.Tyr245Cys and p.Ala356Thr, have a high incidence of cardiac abnormalities. The phenotypic expansion has implications for the clinical diagnosis of CHDSKM in patients with germline ABL1 variants.     

Choline Acetyltransferase Mutations Causing Congenital Myasthenic Syndrome: Molecular Findings and Genotype–Phenotype Correlations

Choline acetyltransferase catalyzes the synthesis of acetylcholine at cholinergic nerves. Mutations in human CHAT cause a congenital myasthenic syndrome due to impaired synthesis of ACh; this severe variant of the disease is frequently associated with unexpected episodes of potentially fatal apnea. The severity of this condition varies remarkably, and the molecular factors determining this variability are poorly understood. Furthermore, genotype–phenotype correlations have been difficult to establish in patients with biallelic mutations. We analyzed the protein expression of phosphorylated ChAT of seven CHAT mutations, p.Val136Met, p.Arg207His, p.Arg186Trp, p.Val194Leu, p.Pro211Ala, p.Arg566Cys, and p.Ser694Cys, in HEK‐293 cells to phosphorylated ChAT, determined their enzyme kinetics and thermal stability, and examined their structural changes. Three mutations, p.Arg207His, p.Arg186Trp, and p.Arg566Cys, are novel, and p.Val136Met and p.Arg207His are homozygous in three families and associated with severe disease. The characterization of mutants showed a decrease in the overall catalytic efficiency of ChAT; in particular, those located near the active‐site tunnel produced the most seriously disruptive phenotypic effects. On the other hand, p.Val136Met, which is located far from both active and substrate‐binding sites, produced the most drastic reduction of ChAT expression. Overall, CHAT mutations producing low enzyme expression and severe kinetic effects are associated with the most severe phenotypes.     

Whole‐genome sequencing in patients with ciliopathies uncovers a novel recurrent tandem duplication in IFT140

Ciliopathies represent a wide spectrum of rare diseases with overlapping phenotypes and a high genetic heterogeneity. Among those, IFT140 is implicated in a variety of phenotypes ranging from isolated retinis pigmentosa to more syndromic cases. Using whole‐genome sequencing in patients with uncharacterized ciliopathies, we identified a novel recurrent tandem duplication of exon 27–30 (6.7 kb) in IFT140, c.3454‐488_4182+2588dup p.(Tyr1152_Thr1394dup), missed by whole‐exome sequencing. Pathogenicity of the mutation was assessed on the patients’ skin fibroblasts. Several hundreds of patients with a ciliopathy phenotype were screened and biallelic mutations were identified in 11 families representing 12 pathogenic variants of which seven are novel. Among those unrelated families especially with a Mainzer‐Saldino syndrome, eight carried the same tandem duplication (two at the homozygous state and six at the heterozygous state). In conclusion, we demonstrated the implication of structural variations in IFT140‐related diseases expanding its mutation spectrum. We also provide evidences for a unique genomic event mediated by an Alu–Alu recombination occurring on a shared haplotype. We confirm that whole‐genome sequencing can be instrumental in the ability to detect structural variants for genomic disorders.     

Second family provides further evidence for causation of Steel syndrome by biallelic mutations in COL27A1

Steel syndrome is a rare disorder of the skeleton characterized by facial dysmorphism, short stature, carpal coalition, dislocated radial heads, bilateral hip dislocation and vertical talus. Homozygous variants in COL27A1 were reported in an extending family from Puerto Rico. Here, we report a 5‐year‐old girl from a non‐consanguineous family with facial dysmorphism, short stature, carpal coalition, dislocation of radial heads, bilateral hip dislocation, scoliosis and vertical talus. Exome sequencing identified 2 novel compound heterozygous variants c.521_528del (p.(Cys174Serfs*34)) and c.2119C>T (p.(Arg707*)) in COL27A1 in this child and the parents were heterozygous carriers. We hence report the second molecularly proven case of Steel syndrome and the first case to be reported among non‐Puerto Rican population. Our report further validates the role of COL27A1 mutations in causation of Steel syndrome.