A Benchmark of In-House Homologous Recombination Repair Deficiency Testing Solutions for High-Grade Serous Ovarian Cancer Diagnosis.

Homologous recombination deficiency (HRD) has become an important prognostic and predictive biomarker for patients with high-grade serous ovarian cancer who may benefit from poly-ADP ribose polymerase inhibitors (PARPi) and platinum-based therapies. HRD testing provides relevant information to personalize patients' treatment options and has been progressively incorporated into diagnostic laboratories. Here, we assessed the performance of an in-house HRD testing system deployable in a diagnostic clinical setting, comparing results from two commercially available next-generation sequencing (NGS)-based tumor tests (SOPHiA DDMTM HRD Solution and AmoyDx® (HRD Focus Panel)) with the reference assay from Myriad MyChoice® (CDx). A total of 85 ovarian cancer samples were subject to HRD testing. An overall strong correlation was observed across the three assays evaluated, regardless of the different underlying methods employed to assess genomic instability, with the highest pairwise correlation between Myriad and SOPHiA (R = 0.87, p-value = 3.39 × 10-19). The comparison of the assigned HRD status to the reference Myriad's test revealed a positive predictive value (PPV) and negative predictive value (NPV) of 90.9% and 96.3% for SOPHiA's test, while AmoyDx's test achieved 75% PPV and 100% NPV. This is the largest HRD testing evaluation using different methodologies and provides a clear picture of the robustness of NGS-based tests currently offered in the market. Our data shows that the implementation of in-house HRD testing in diagnostic laboratories is technically feasible and can be reliably performed with commercial assays. Also, the turnaround time is compatible with clinical needs, making it an ideal alternative to offer to a broader number of patients while maintaining high-quality standards at more accessible price tiers.

Biallelic variants in DNA2 cause poikiloderma with congenital cataracts and severe growth failure reminiscent of Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is a rare, heterogeneous autosomal recessive genodermatosis, with poikiloderma as its hallmark. It is classified into two types: type I, with biallelic variants in ANAPC1 and juvenile cataracts, and type II, with biallelic variants in RECQL4, increased cancer risk and no cataracts. We report on six Brazilian probands and two siblings of Swiss/Portuguese ancestry presenting with severe short stature, widespread poikiloderma and congenital ocular anomalies. Genomic and functional analysis revealed compound heterozygosis for a deep intronic splicing variant in trans with loss of function variants in DNA2, with reduction of the protein levels and impaired DNA double-strand break repair. The intronic variant is shared by all patients, as well as the Portuguese father of the European siblings, indicating a probable founder effect. Biallelic variants in DNA2 were previously associated with microcephalic osteodysplastic primordial dwarfism. Although the individuals reported here present a similar growth pattern, the presence of poikiloderma and ocular anomalies is unique. Thus, we have broadened the phenotypical spectrum of DNA2 mutations, incorporating clinical characteristics of RTS. Although a clear genotype-phenotype correlation cannot be definitively established at this moment, we speculate that the residual activity of the splicing variant allele could be responsible for the distinct manifestations of DNA2-related syndromes.

A dopamine receptor D2 genetic polymorphism associated with transition to mental disorders in a cohort of individuals with at-risk mental state for psychosis

Objectives: To test the association of 45 single nucleotide polymorphisms (SNPs) with transition to psychiatric disorders in a cohort of individuals at ultrahigh risk (UHR) mental state for psychosis. Methods: Through general population screening, 88 non-help-seeking UHR subjects and 130 healthy control individuals were genotyped for 45 SNPs related to psychosis. They were followed for a mean of 2.5 years, and conversion to psychotic and to general psychiatric disorders was assessed. Genotype frequencies between controls, converters, and non-converters were analyzed. Results: There were no differences in sociodemographics between controls and UHR. Also, UHR converters and non-converters had no differences in their baseline symptoms scores. The dopamine receptor D2 gene (DRD2) SNP rs6277 was significantly more common among UHR who transitioned to psychosis (p < 0.001) and to UHR who transitioned to any psychiatric disorders (p = 0.001) when compared to UHR who did not transition. The rs6277 T allele was related to psychiatric morbidity in a dose-response fashion, being significantly more frequent in UHR converters than UHR non-converters and control subjects (p = 0.003). Conclusion: Our findings suggest that rs6277 could potentially constitute a genetic marker of transition to psychiatric disorders in subjects with at-risk mental states, warranting further investigation in larger samples.

Atypical, severe hypertrophic cardiomyopathy in a newborn presenting Noonan syndrome harboring a recurrent heterozygous MRAS variant.

Noonan syndrome (NS) is a Mendelian phenotype, member of a group of disorders sharing neurocardiofaciocutaneous involvement, known as RASopathies, caused by germline variants in genes coding for components of the RAS/MAPK signaling pathway. Recently, a novel gene of the RAS family (MRAS) was reported to be associated with NS in five children, all of them presenting, among the cardinal features of NS, the same cardiac finding, hypertrophic cardiomyopathy (HCM). We report on a 2-month-old infant boy also presenting this cardiac anomaly that evolved to a fatal outcome after a surgical myectomy. In addition, a thick walled left ventricle apical aneurysm, rarely described in NS, was also disclosed. Next-generation sequencing revealed a missense, previously reported variant in MRAS (p.Thr68Ile). This report reinforces the high frequency of HCM among individuals harboring MRAS variants, contrasting to the 20% overall prevalence of this cardiac anomaly in NS. Thus, these preliminary data suggest that variants in MRAS per se are high risk factors for the development of an early, severe HCM, mostly of them with left ventricle outflow tract obstruction, with poor prognosis. Because of the severity of the cardiac involvement, other clinical findings could not be addressed in detail. Therefore, long-term follow-up of these individuals and further descriptions are required to fully understand the complete phenotypic spectrum of NS associated with MRAS germline variants, including if these individuals present an increased risk for cancer.

Bi-allelic CSF1R Mutations Cause Skeletal Dysplasia of Dysosteosclerosis-Pyle Disease Spectrum and Degenerative Encephalopathy with Brain Malformation.

Colony stimulating factor 1 receptor (CSF1R) plays key roles in regulating development and function of the monocyte/macrophage lineage, including microglia and osteoclasts. Mono-allelic mutations of CSF1R are known to cause hereditary diffuse leukoencephalopathy with spheroids (HDLS), an adult-onset progressive neurodegenerative disorder. Here, we report seven affected individuals from three unrelated families who had bi-allelic CSF1R mutations. In addition to early-onset HDLS-like neurological disorders, they had brain malformations and skeletal dysplasia compatible to dysosteosclerosis (DOS) or Pyle disease. We identified five CSF1R mutations that were homozygous or compound heterozygous in these affected individuals. Two of them were deep intronic mutations resulting in abnormal inclusion of intron sequences in the mRNA. Compared with Csf1r-null mice, the skeletal and neural phenotypes of the affected individuals appeared milder and variable, suggesting that at least one of the mutations in each affected individual is hypomorphic. Our results characterized a unique human skeletal phenotype caused by CSF1R deficiency and implied that bi-allelic CSF1R mutations cause a spectrum of neurological and skeletal disorders, probably depending on the residual CSF1R function.

Diagnosis of Atelosteogenesis Type I suggested by Fetal Ultrasonography and Atypical Paternal Phenotype with Mosaicism.

Atelosteogenesis type I (AOI) is an autosomal dominant skeletal dysplasia caused by mutations in the filaminB (FLNB) gene with classic and well-recognizable clinical findings. However, parents affected with a mild phenotype, probably with somatic mosaicism, can generate offspring with a much more severe phenotype of AOI. In the present report, we describe a female newborn with classic AOI leading to early neonatal death, whose diagnostic was based on prenatal radiological findings and on the physical examination of the father. Since her father had limb deformities and corporal asymmetry, suggesting somatic mosaicism, his biological samples were analyzed through a gene panel for skeletal dysplasias. A missense mutation not previously described in the literature was detected in the FLNB gene, affecting ∼ 20% of the evaluated cells and, therefore, confirming the diagnosis of mosaic AOI in the father. The molecular analysis of the father was crucial to suggest the diagnosis of AOI in the newborn, since she died early and there were no biological samples available.

A atelosteogênese tipo I (AOI) é uma displasia esquelética autossômica dominante causada por mutações no gene filamina B (FLNB) com achados clínicos clássicos e bem reconhecíveis. No entanto, pais afetados com um fenótipo mais leve, provavelmente com mosaicismo somático, podem gerar uma prole com um fenótipo muito mais grave de AOI. No presente relato, descrevemos um recém-nascido do sexo feminino com AOI clássica, que levou à morte neonatal precoce, e cujo diagnóstico foi baseado em achados radiológicos pré-natais e no exame físico de seu genitor. Como o genitor apresentava deformidades em membros e assimetria corporal, que sugeriam mosaicismo somático, suas amostras biológicas foram analisadas por meio de um painel de genes para displasias esqueléticas. Uma mutação missense, não descrita anteriormente na literatura, foi detectada no gene FLNB, afetando ∼ 20% das células avaliadas, e, portanto, confirmando o diagnóstico de AOI em mosaico no genitor. A análise molecular realizada no genitor foi fundamental para sugerir o diagnóstico de AOI na recém-nascida, uma vez que esta morreu precocemente, e não havia amostras biológicas disponíveis.

Natural history of 39 patients with Achondroplasia.

OBJECTIVES: To characterize the natural history of 39 achondroplastic patients diagnosed by clinical, radiological and molecular assessments. METHODS: Observational and retrospective study of 39 patients who were attended at a public tertiary level hospital between 1995 and 2016. RESULTS: Diagnosis was made prenatally in 11 patients, at birth in 9 patients and within the first year of life in 13 patients. The most prevalent clinical findings were short stature, high forehead, trident hands, genu varum and macrocephaly. The most prevalent radiographic findings were rhizomelic shortening of the long bones and narrowing of the interpediculate distance of the caudal spine. There was motor developmental delay in 18 patients and speech delay in 16 patients. The most common clinical intercurrences were middle ear dysfunction, sleep apnea, limb pain and obesity from 2 to 9 years of age. One patient was large for the gestational age but did not develop obesity. One patient developed hydrocephalus at 10 years old. The current age of the patients varies from 15 months to 36 years. The molecular study performed by Sanger sequencing of the common heterozygous mutation 1138G>A in FGFR3 was positive in all patients. Four cases were inherited, and 35 were sporadic (paternal age from 19 to 66 years). CONCLUSIONS: The diagnoses were made early based on clinical and radiographic findings. All cases were confirmed molecularly. Despite presenting a benign course, it is necessary to establish a systematic protocol for the surveillance of these patients due to the common clinical intercurrences.

Mutation analysis of SLC26A4 (Pendrin) gene in a Brazilian sample of hearing-impaired subjects.

BACKGROUND: Mutations in the SLC26A4 gene are associated with Pendred syndrome and autosomal recessive non-syndromic deafness (DFNB4). Both disorders have similar audiologic characteristics: bilateral hearing loss, often severe or profound, which may be associated with abnormalities of the inner ear, such as dilatation of the vestibular aqueduct or Mondini dysplasia. But, in Pendred syndrome (OMIM #274600), with autosomal recessive inheritance, besides congenital sensorineural deafness, goiter or thyroid dysfunctions are frequently present. The aim of this study was to determine whether mutations in SLC26A4 are a frequent cause of hereditary deafness in Brazilian patients. METHODS: Microsatellite haplotypes linked to SLC26A4 were investigated in 68 families presenting autosomal recessive non-syndromic deafness. In the probands of the 16 families presenting segregation consistent with linkage to SLC26A4, Sanger sequencing of the 20 coding exons was performed. In an additional sample of 15 individuals with suspected Pendred syndrome, because of the presence of hypothyroidism or cochleovestibular malformations, the SLC26A4 gene coding region was also sequenced. RESULTS: In two of the 16 families with indication of linkage to SLC26A4, the probands were found to be compound heterozygotes for probably pathogenic different mutations: three novel (c.1003 T > G (p. F335 V), c.1553G > A (p.W518X), c.2235 + 2 T > C (IVS19 + 2 T > C), and one already described, c.84C > A (p.S28R). Two of the 15 individuals with suspected Pendred syndrome because of hypothyreoidism or cochleovestibular malformations were monoallelic for likely pathogenic mutations: a splice mutation (IVS7 + 2 T > C) and the previously described c.1246A > C (p.T416P). Pathogenic copy number variations were excluded in the monoallelic cases and in those with normal results after Sanger sequencing. Additional mutations in the SLC26A4 gene or other definite molecular cause for deafness were not identified in the monoallelic patients, after exome sequencing. CONCLUSIONS: Biallelic pathogenic mutations in SLC26A4 explained ~ 3% of cases selected because of autosomal recessive deafness. Monoallelic mutations were present in ~ 13% of isolated cases of deafness with cochleovestibular malformations or suspected Pendred syndrome. These data reinforce the importance of mutation screening of SLC26A4 in Brazilian subjects and highlight the elevated frequency of monoallelic patients.

Expanding the Clinical Spectrum of Phenotypes Caused by Pathogenic Variants in PLOD2.

Osteogenesis imperfecta (OI) is a strikingly heterogeneous group of disorders with a broad range of phenotypic variations. It is also one of the differential diagnoses in bent bone dysplasias along with campomelic dysplasia and thanatophoric dysplasia and can usually be distinguished by decreased bone mineralization and bone fractures. Bent bone dysplasias also include syndromes such as kyphomelic dysplasia (MIM:211350) and mesomelic dysplasia Kozlowski-Reardon (MIM249710), both of which have been under debate regarding whether or not they are a real entity or simply a phenotypic manifestation of another dysplasia including OI. Bruck syndrome type 2 (BRKS2; MIM:609220) is a rare form of autosomal recessive OI caused by biallelic PLOD2 variants and is associated with congenital joint contractures with pterygia. In this report, we present six patients from four families with novel PLOD2 variants. All cases had multiple fractures. Other features ranged from prenatal lethal severe angulation of the long bones as in kyphomelic dysplasia and mesomelic dysplasia Kozlowski-Reardon through classical Bruck syndrome to moderate OI with normal joints. Two siblings with a kyphomelic dysplasia-like phenotype who were stillborn had compound heterozygous variants in PLOD2 (p.Asp585Val and p.Ser166*). One infant who succumbed at age 4 months had a bent bone phenotype phenotypically like skeletal dysplasia Kozlowski-Reardon (with mesomelic shortening, camptodactyly, retrognathia, cleft palate, skin dimples, but also with fractures). He was homozygous for the nonsense variant (p.Trp561*). Two siblings had various degrees of Bruck syndrome caused by the homozygous missense variant, p.His687Arg. Furthermore a boy with a clinical presentation of moderate OI had a possibly pathogenic homozygous variant p.Trp588Cys. Our experience of six patients with biallelic pathogenic variants in PLOD2 expands the phenotypic spectrum in the PLOD2-related phenotypes. © 2017 American Society for Bone and Mineral Research.

Natural history of 39 patients with Achondroplasia

OBJECTIVES: To characterize the natural history of 39 achondroplastic patients diagnosed by clinical, radiological and molecular assessments. METHODS: Observational and retrospective study of 39 patients who were attended at a public tertiary level hospital between 1995 and 2016. RESULTS: Diagnosis was made prenatally in 11 patients, at birth in 9 patients and within the first year of life in 13 patients. The most prevalent clinical findings were short stature, high forehead, trident hands, genu varum and macrocephaly. The most prevalent radiographic findings were rhizomelic shortening of the long bones and narrowing of the interpediculate distance of the caudal spine. There was motor developmental delay in 18 patients and speech delay in 16 patients. The most common clinical intercurrences were middle ear dysfunction, sleep apnea, limb pain and obesity from 2 to 9 years of age. One patient was large for the gestational age but did not develop obesity. One patient developed hydrocephalus at 10 years old. The current age of the patients varies from 15 months to 36 years. The molecular study performed by Sanger sequencing of the common heterozygous mutation 1138G>A in FGFR3 was positive in all patients. Four cases were inherited, and 35 were sporadic (paternal age from 19 to 66 years). CONCLUSIONS: The diagnoses were made early based on clinical and radiographic findings. All cases were confirmed molecularly. Despite presenting a benign course, it is necessary to establish a systematic protocol for the surveillance of these patients due to the common clinical intercurrences.

Post-mortem cytogenomic investigations in patients with congenital malformations.

Congenital anomalies are the second highest cause of infant deaths, and, in most cases, diagnosis is a challenge. In this study, we characterize patterns of DNA copy number aberrations in different samples of post-mortem tissues from patients with congenital malformations. Twenty-eight patients undergoing autopsy were cytogenomically evaluated using several methods, specifically, Multiplex Ligation-dependent Probe Amplification (MLPA), microsatellite marker analysis with a MiniFiler kit, FISH, a cytogenomic array technique and bidirectional Sanger sequencing, which were performed on samples of different tissues (brain, heart, liver, skin and diaphragm) preserved in RNAlater, in formaldehyde or by paraffin-embedding. The results identified 13 patients with pathogenic copy number variations (CNVs). Of these, eight presented aneuploidies involving chromosomes 13, 18, 21, X and Y (two presented inter- and intra-tissue mosaicism). In addition, other abnormalities were found, including duplication of the TYMS gene (18p11.32); deletion of the CHL1 gene (3p26.3); deletion of the HIC1 gene (17p13.3); and deletion of the TOM1L2 gene (17p11.2). One patient had a pathogenic missense mutation of g.8535C>G (c.746C>G) in exon 7 of the FGFR3 gene consistent with Thanatophoric Dysplasia type I. Cytogenomic techniques were reliable for the analysis of autopsy material and allowed the identification of inter- and intra-tissue mosaicism and a better understanding of the pathogenesis of congenital malformations.

Nutritional aspects of Noonan syndrome and Noonan-related disorders.

Rasopathies are a group of rare disorders characterized by neurocardiofaciocutaneous involvement, and caused by mutations in several genes of the RAS/MAPK pathway. In the present study, we characterized growth parameters, body composition, and nutritional aspects of children and adults (n = 62) affected by these disorders, mainly Noonan syndrome, using an indirect method-anthropometry-and a 24-hr recall questionnaire. The growth parameters in our cohort showed short stature, especially in individuals with RAF1 and SHOC2 mutations, lower obesity rates compared to the control population, and BMI scores highest in individuals with BRAF mutations and lowest in individuals with SHOC2. Body composition showed a compromise in the upper arm muscle circumference, with a statistically significant difference in the z-score of triceps skinfold (P = 0.0204) and upper arm fat area (P = 0.0388) between BRAF and SHOC2 groups and in the z-score of triceps skinfold between RAF1 and SHOC2 (P = 0.0218). The pattern of macronutrient consumption was similar to the control population. Our study is the first to address body composition in RASopathy individuals and the data indicate a compromise not only in adipose tissue, but also in muscle mass. Studies using different techniques, such as dual-energy X-ray absorptiometry or imaging studies, which give a more precise delineation of fat and non-fat mass, are required to confirm our results, ultimately causing an impact on management strategies. © 2016 Wiley Periodicals, Inc.

Rare Variants in the Epithelial Cadherin Gene Underlying the Genetic Etiology of Nonsyndromic Cleft Lip with or without Cleft Palate.

Nonsyndromic orofacial cleft (NSOFC) is a complex disease of still unclear genetic etiology. To investigate the contribution of rare epithelial cadherin (CDH1) gene variants to NSOFC, we target sequenced 221 probands. Candidate variants were evaluated via in vitro, in silico, or segregation analyses. Three probably pathogenic variants (c.760G>A [p.Asp254Asn], c.1023T>G [p.Tyr341*], and c.2351G>A [p.Arg784His]) segregated according to autosomal dominant inheritance in four nonsyndromic cleft lip with or without cleft palate (NSCL/P) families (Lod score: 5.8 at θ = 0; 47% penetrance). A fourth possibly pathogenic variant (c.387+5G>A) was also found, but further functional analyses are needed (overall prevalence of CDH1 candidate variants: 2%; 15.4% among familial cases). CDH1 mutational burden was higher among probands from familial cases when compared to that of controls (P = 0.002). We concluded that CDH1 contributes to NSCL/P with mainly rare, moderately penetrant variants, and CDH1 haploinsufficiency is the likely etiological mechanism.