Multiple malformation syndromes (MMS) belong to a group of genetic disorders characterised by neurodevelopmental anomalies and congenital malformations. Here we explore for the first time the genetic ...aetiology of MMS using whole-exome sequencing (WES) in undiagnosed patients from the Greek-Cypriot population after prior extensive diagnostics workup including karyotype and array-CGH. A total of 100 individuals (37 affected), from 32 families were recruited and family-based WES was applied to detect causative single-nucleotide variants (SNVs) and indels. A genetic diagnosis was reported for 16 MMS patients (43.2%), with 10/17 (58.8%) of the findings being novel. All autosomal dominant findings occurred de novo. Functional studies were also performed to elucidate the molecular mechanism relevant to the abnormal phenotypes, in cases where the clinical significance of the findings was unclear. The 17 variants identified in our cohort were located in 14 genes (PCNT, UBE3A, KAT6A, SPR, POMGNT1, PIEZO2, PXDN, KDM6A, PHIP, HECW2, TFAP2A, CNOT3, AGTPBP1 and GAMT). This study has highlighted the efficacy of WES through the high detection rate (43.2%) achieved for a challenging category of undiagnosed patients with MMS compared to other conventional diagnostic testing methods (10-20% for array-CGH and ~3% for G-banding karyotype analysis). As a result, family-based WES could potentially be considered as a first-tier cost effective diagnostic test for patients with MMS that facilitates better patient management, prognosis and offer accurate recurrence risks to the families.
Background
Boucher Neuhäuser Syndrome (BNS) is a rare disease with autosomal recessive inheritance defined by the classical triad; early-onset ataxia, hypogonadism and chorioretinal dystrophy.
Case ...presentation
We present two siblings diagnosed with BNS at midlife, identified with homozygous state of a novel PNPLA6 missense mutation. One healthy sibling and the mother were heterozygous carriers of the mutation. The proband presented with the classical triad and the other sibling presented with visual problems at first. The proband was referred to our department by a private Neurologist, in early adulthood, because of hypogonadism, cerebellar ataxia, axonal neuropathy, and chorioretinal dystrophy for further evaluation. The sibling was referred to our department for evaluation, at childhood, due to visual problems. Later, the patient displayed the triad of ataxia, hypogonadotropic hypogonadism, and chorioretinal dystrophy. The unusual medical history of the two siblings led to further examinations and eventually the diagnosis of the first BNS cases in Cyprus. WES-based ataxia in silico gene panel analysis revealed 15 genetic variants and further filtering analysis revealed the PNPLA6 c.3323G > A variant. Segregation analysis in the family with Sanger sequencing confirmed the PNPLA6 homozygous variant c.3323G > A, p.Arg1108Gln in exon 29.
Conclusions
This highlights the importance of considering rare inherited causes of visual loss, spinocerebellar ataxia, or/and HH in a neurology clinic and the significant role of genetic sequencing in the diagnostic process.
The study aimed to identify the pathogenic status of p.Gln319Ter (NM_000500.7: c.955C>T) variant when inherited in a single
gene (bimodular RCCX haplotype) and to discriminate between a non-causing ...congenital adrenal hyperplasia (CAH) allele when inherited in a duplicated and functional
gene context (trimodular RCCX haplotype).
38 females and 8 males with hyperandrogenemia, previously screened by sequencing and identified as carriers for the pathogenic p.Gln319Ter, were herein tested by multiplex ligation-dependent probe amplification (MLPA) and a real-time PCR Copy number Variation (CNV) assay.
Both MLPA and real-time PCR CNV analyses confirmed a bimodular and pathogenic RCCX haplotype with a single
in 19/46 (41.30%) p.Gln319Ter carriers and who in parallel all shared elevated 17-OHP levels. The remaining 27 individuals that also carried the p.Gln319Ter exhibited low 17-OHP levels as a result of their carriership of a duplicated
with a trimodular RCCX haplotype. Interestingly, all of these individuals also carried in linkage disequilibrium with p.Gln319Ter two single nucleotide polymorphisms, the c.293-79G>A (
) in intron 2 and the c.*12C>T (
) in the 3'-UTR. Therefore, these variants can be used to distinguish between pathogenic and non-pathogenic genomic contexts of the c.955T (p.Gln319) in the genetic diagnosis of congenital adrenal hyperplasia (CAH).
The employed methodologies identified a considerable number of individuals with non-pathogenic p.Gln319Ter from the individuals that typically carry the pathogenic p.Gln319Ter in a single
. Therefore, it is extremely important the detection of such haplotypes for the prenatal diagnosis, treatment and genetic counseling in patients with CAH.
Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease caused by Gonadotropin-Releasing Hormone (GnRH) deficiency. So far a limited number of variants in several genes have been ...associated with the pathogenesis of the disease. In this original research and review manuscript the retrospective analysis of known variants in
(
, and
genes is described, along with novel variants identified in patients with CHH by the present study.
Seven GnRH deficient unrelated Cypriot patients underwent whole exome sequencing (WES) by Next Generation Sequencing (NGS). The identified novel variants were initially examined by
computational algorithms and structural analysis of their predicted pathogenicity at the protein level was confirmed.
In four non-related GnRH males, a novel X-linked pathogenic variant in
gene, two novel autosomal dominant (AD) probably pathogenic variants in
and
genes and one rare AD probably pathogenic variant in
gene were identified. A rare autosomal recessive (AR) variant in the
gene was identified in homozygosity in a female patient, whilst two other male patients were also, respectively, found to carry novel or previously reported rare pathogenic variants in more than one genes;
/
and
.
This report embraces the description of novel and previously reported rare pathogenic variants in a series of genes known to be implicated in the biological development of CHH. Notably, patients with CHH can harbor pathogenic rare variants in more than one gene which raises the hypothesis of locus-locus interactions providing evidence for digenic inheritance. The identification of such aberrations by NGS can be very informative for the management and future planning of these patients.
The neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of autosomal recessive lysosomal storage disorders that are characterized by neurodegeneration, progressive ...cognitive decline, motor impairment, ataxia, loss of vision, seizures, and premature death. To date, pathogenic variants in more than 13 genes have been associated with NCLs.
CLN6
encodes an endoplasmic reticulum non-glycosylated transmembrane protein, which is involved in lysosomal acidification. Mutations in
CLN6
cause late-infantile juvenile NCL (JNCL) adult-onset NCL, and Kufs disease. Members from two available families with JNCL were clinically evaluated, and samples were collected from consenting individuals. The molecular investigation was performed by whole-exome sequencing, Sanger sequencing, and family segregation analysis. Furthermore,
in silico
prediction analysis and structural modeling of the identified
CLN6
variants were performed. We report clinical and genetic findings of three patients from two Greek-Cypriot families (families 915 and 926) with JNCL. All patients were males, and the first symptoms appeared at the age of 6 years. The proband of family 926 presented with loss of motor abilities, ataxia, spasticity, seizure, and epilepsy. The proband of family 915 had ataxia, spasticity, dysarthria, dystonia, and intellectual disability. Both probands did not show initial signs of vision and/or hearing loss. Molecular analysis of family 926 revealed two
CLN6
biallelic variants: the novel,
de novo
p.Tyr295Cys and the known p.Arg136His variants. In family 915, both patients were homozygous for the p.Arg136His
CLN6
variant. Prediction analysis of the two
CLN6
variants characterized them as probably damaging and disease-causing. Structural modeling of the variants predicted that they probably cause protein structural differentiation. In conclusion, we describe two unrelated Cypriot families with JNCL. Both families had variants in the
CLN6
gene; however, they presented with slightly different symptoms, and notably none of the patients has loss of vision.
In silico
prediction and structural analyses indicate that both variants are most likely pathogenic.
Abstract
Background
Specialized clinical care for cystic fibrosis (CF) in Cyprus, a small island country, has been implemented since the 1990s. However, only recently, a national CF patient registry ...has been established for the systematic recording of patients’ data. In this study, we aim to present data on the epidemiological, genotypic and phenotypic features of CF patients in the country from the most recent data collection in 2019, with particular emphasis on notable rare or unique cases.
Results
Overall, data from 52 patients are presented, 5 of whom have deceased and 13 have been lost to follow-up in previous years. The mean age at diagnosis was 7.2 ± 12.3 years, and the mean age of 34 alive patients by the end of 2019 was 22.6 ± 13.2 years. Patients most commonly presented at diagnosis with acute or persistent respiratory symptoms (46.2%), failure to thrive or malnutrition (40.4%), and dehydration or electrolyte imbalance (32.7%). Sweat chloride levels were diagnostic (above 60 mmol/L) in 81.8% of examined patients. The most common identified mutation was p.Phe508del (F508del) (45.2%), followed by p.Leu346Pro (L346P) (6.7%), a mutation detected solely in individuals of Cypriot descent. The mean BMI and FEV
1
z-scores were 0.2 ± 1.3 and − 2.1 ± 1.7 across all age groups, respectively, whereas chronic
Pseudomonas aeruginosa
colonization was noted in 26.9% of patients. The majority of patients (74.5%) were eligible to receive at least one of the available CFTR modulator therapies. In 25% of patients we recovered rare or unique genotypic profiles, including the endemic p.Leu346Pro (L346P), the rare CFTR-dup2, the co-segregated c.4200_4201delTG/c.489 + 3A > G, and the polymorphism p.Ser877Ala.
Conclusions
CF patient registries are particularly important in small or isolated populations, such as in Cyprus, with rare or unique disease cases. Their operation is necessary for the optimization of clinical care provided to CF patients, enabling their majority to benefit from evolving advances in precision medicine.
Oculodentodigital dysplasia syndrome is associated with numerous pathogenic variants in
, the gene encoding connexin43 gap junction protein. A novel in-frame deletion (p.Lys134del) was found in our ...clinic. The patient showed all the typical dysmorphic features of the syndrome. The functional consequences of this variant were also studied in an
system. Cells expressed significantly less number of gap junction plaques with a great number of them retained intracellularly.
Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder characterized by the development of multiple, circumscript and usually symmetric bony protuberances called ...osteochondromas. Most HME are caused by EXT1 and EXT2 loss of function mutations. Most pathogenic mutations are nonsense followed by missense mutations and deletions.
Here we report on a patient with a rare and complex genotype resulting in a typical HME phenotype. Initial point mutation screening in EXT1 and EXT2 genes by Sanger sequencing did not reveal any pathogenic variants. The patient along with the healthy parents was subsequently referred for karyotype and array-Comparative Genomic Hybridization (CGH) analyses. Chromosomal analysis revealed two independent de novo apparently balanced rearrangements: a balanced translocation between the long arms of chromosomes 2 and 3 at breakpoints 2q22 and 3q13.2 and a pericentric inversion with breakpoints at 8p23.1q24.1. Both breakpoints were confirmed by Fluorescence In Situ Hybridization (FISH). Subsequently, array-CGH revealed a novel heterozygous deletion within the EXT1 gene at one of the inversion breakpoints, rendering the inversion unbalanced. The mode of inheritance, as well as the size of the deletion were further investigated by Quantitative Real-time PCR (qPCR), defining the deletion as de novo and of 3.1 kb in size, removing exon 10 of EXT1. The inversion in combination with the 8p23.1 deletion most likely abolishes the transcription of EXT1 downstream of exon 10 hence resulting in a truncated protein.
The identification of a rare and novel genetic cause of HME, highlights the importance of additional comprehensive investigation of patients with typical clinical manifestations, even when EXT1 and EXT2 mutation analysis is negative.
Steel syndrome is an autosomal recessive disorder that primarily affects the skeletal system causing a variety of manifestations. Sixteen individuals with Steel syndrome, mainly Puerto Ricans ...(11/16), were previously reported to carry bi-allelic mutations in the COL27A1 gene. Here, we present the first patient with Steel syndrome in Europe and the sixth non-Puerto Rican carrying a novel homozygous mutation in COL27A1. The patient is a 4-year-old boy born to non-consanguineous healthy parents, with dysmorphic facial features, absent hip ossification centres, external rotation of both feet, relatively short stature, mild skin syndactyly, short mid phalanges and bilateral sensorineural hearing loss. Whole exome sequencing (WES) revealed a novel homozygous missense variant p.(Gly802Glu) in COL27A1. The homozygous mutation was confirmed by Sanger sequencing in the proband and carrier status was confirmed in both parents and his unaffected sibling. According to online and in-house minor allele frequency (MAF) databases, this is the first COL27A1 mutation reported in the European population. Additional screening of healthy Greek-Cypriot individuals was thus performed, which did not reveal any additional carriers in the population for the variant in question.
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