Exome sequencing was performed using the Twist Comprehensive Exome v1 Kit. Following genomic DNA (gDNA) extraction and concentration normalization, tagmentation, amplification, and ligation of unique sample markers were carried out. Libraries were enriched via hybridization with biotinylated probes targeting exonic regions, followed by final amplification, purification, and quality assessment, requiring a concentration above 3 ng/µl and a fragment size of ∼330 bp. Sequencing was conducted on the NovaSeq6000 (Illumina), and data were processed using Varsome Clinical with the GRCh37/Hg19 reference genome. Variant interpretation followed ACMG guidelines (13) and ClinGen recommendations. The minimum quality metrics for case analysis were 95% total coverage and more than 100× depth. In addition, all identified variants had at least 50 reads depth and an allele fraction greater than 30%.

For aCGH processing, genomic DNA was first extracted from a peripheral blood sample using the protocol suggested by the manufacturer company, Agilent®. DNA digestion was performed using Alu I and RSA I enzymes from both patient and reference samples, followed by fluorochrome labelling of patient DNA and reference DNA (male and female control) using Cy5 and Cy3 fluorochromes. The DNA was then purified on columns and quantified to ensure concentration, yield and specific activity, parameters necessary to compare patient and reference DNA. Hybridisation was then performed using the Agilent® SurePrint G3 Human ICGH+SNP 4 × 180 K array. Finally, the scan was performed using the SureScan® platform and data was acquired, quality parameters were evaluated and results were analyzed using Agilent CytoGenomics v5R software. Analysis was performed using available databases: DECIPHER, DGV (Database of Genomic Variants), ClinVar-National Center for Biotechnology and the American College of Genetic Medicine guidelines for interpretation and reporting of copy number variants (14).

A statistical descriptive analysis was performed to determine the number of patients who underwent at least one of the two types of molecular genetic testing: WES (single and trio) and aCGH. Patients were stratified according to sex, and it was determined how many male and female patients were referred for each of these tests. In addition, the results of each test were classified into three categories: positive, negative, and variants of uncertain significance (VUS).The overall diagnostic yield of the genetic tests was determined by considering the proportion of positive results (those with pathogenic or likely pathogenic variants explaining the patient's phenotype) compared to VUS or negative results.

A variant statistical analysis was also performed including all variants (SNVs, deletions, insertions and CNVs) detected with both tests. This analysis allowed us to identify those variants that occurred more frequently in the population with positive results, novel variants that have not been reported in the databases and the chromosomes and the most frequently altered chromosomes.

On the other hand, due to the sample size and the phenotypic heterogeneity of the patients, Fisher's exact test was used to statistically assess whether there was a significant association between the presence of specific phenotypes and the results of the molecular studies (15). This was done by analyzing the proportion of the event in the group of interest. For this calculation, only cases with a pathogenic or likely pathogenic variant explaining the patient's phenotype were considered positive. Reports containing variants of uncertain significance (VUS) or no clinically significant variants were classified as negative. The formula is defined as follows:p=(a+ca)(b+db)(na+b).To evaluate the association of the positive results vs. the presence or absence of the phenotypes, odds ratios (OR) with their respective 95% confidence intervals were calculated from the following formula:OR=(a/c)(b/d)95%CI=eLn(OR)±1.96×(1/a)+(1/b)+(1/c)+(1/d).

A phenotypic analysis was performed by extracting the most specific HPO terms from the electronic health record (EHR) of each patient. These specific terms were propagated to the highest possible level term within the “Phenotypic abnormality” term hierarchy of 23 terms according to the method described by Galer et al. (16) in order to group similar phenotypes into the same HPO term, reduce the number of HPO terms to be analyzed, and more clearly visualize the patterns present among patients. In this way, a patient with neurodevelopmental delay (HP:0012758) and a patient with intellectual disability (HP:0001249) would be grouped with the HPO “Abnormality of the nervous system” (HP:0000707). The terms “Abnormality of limbs” (HP:0040064) and “Abnormality of the musculoskeletal system” (HP:0033127) are at the same hierarchical level within “Phenotypic abnormality”, and in turn, share terms within their hierarchies. Therefore, a panel of experts determined under which of the two terms the shared phenotypes should be classified. For example, the term Short metacarpal (HP:0010049) was propagated to “Abnormality of limbs” (HP:0040064).

HPO terms propagated were organized in a heatmap according to their similarity, together with the genetic test results of the patients. Dendrograms for both HPO terms and patients were used to identify clustering patterns. The analysis allowed detection of HPO terms with the highest prevalence of positive results, as well as common phenotypic features among patients with pathogenic results and variants of uncertain significance. These dendrograms were used for the construction of the heatmap; a hierarchical clustering analysis was performed to identify phenotypic patterns and determine similarities among patients based on HPO terms. Clustering techniques were used to group patients and phenotypes with similar characteristics, thereby facilitating the identification of diagnostic patterns. To measure the similarity between patient profiles, Euclidean distance was employed. Additionally, Ward's method, which minimizes variance within each group, was applied to optimize the clustering of patients based on their phenotypic characteristics. The graphical representation allowed the identification of groups of phenotypes and their correlation with the different diagnostic results.

A total of 82 WES tests were performed, with 46.3% (n = 38) conducted on male patients and 53.7% (n = 44) on female patients. The tests included 60 single exomes (73.2%) and 22 trios (26.8%). Of these, 35 (42.7%) yielded negative results, while 25 (30.5%) were positive (n = 15 single; n = 10 trio), confirming pathogenic genetic variants linked to the investigated diseases. Additionally, 22 tests (26.8%) identified variants of uncertain significance (VUS).

Among the positive WES, 5 patients had one or more CNVs, with one patient having both a deletion and a duplication, identifying a total of 6 CNVs (n = 6). 5 CNVs were classified as pathogenic (n = 5) (3 duplications and 2 deletions) and one was likely pathogenic (n = 1) (1 deletion).

Of the 20 SNVs reported in WES, 4 were found that had not been reported in Clinvar or other databases, the description of the novel variants reported is shown in Table 2.

The first patient (P1) was a 14-day-old female with prenatal diagnosis of cerebral cortical malformation, polymicrogyria, colpocephaly and secondary ventriculomegaly, identified by cerebral MRI at 30 weeks of gestation. Five weeks after birth, a trio of WES was performed and a de novo heterozygous variant in the GRIN1 gene (c.1824G>C; p.Trp608Cys) associated with neurodevelopmental disorder with an autosomal dominant inheritance pattern with or without hyperkinetic movements and seizures (MIM#614254) was identified. It has been shown that patients affected by variants in the gene, in addition to seizures and neurodevelopmental disorder, have alterations in radial and tangential neuronal migration (17). The patient had a fatal outcome at three months of age (See Figure 2 for diagnostic timeline).

Patient 2 (P2) was born preterm at 33 weeks of gestation and 25 days old at the time of the study request, with a pediatric dermatology diagnosis at birth of congenital ichthyosiform erythroderma, retrognathia, macroglossia and glossoptosis, who underwent an individual WES 3 weeks after birth. The study identified a possible compound heterozygosity in the ABCA12 gene, consisting of the variants c.4139A>G (p.Asn1380Ser), reported as pathogenic in the Clinvar database (rs28940269) and c.1789del (p.Ser597Leufs*18), which has not been reported in the databases so far. The NMD prediction tool classifies this variant as subject to nonsense-mediated decay degradation. Currently, it has not been possible to study the parents to evaluate the segregation of the variants in each parent, which would allow us to confirm the diagnosis of autosomal recessive congenital ichthyosis 4A (MIM#601277) or autosomal recessive congenital ichthyosis 4B (harlequin) (MIM#242500). Clinically, the patient was managed under the guidance of the dermatology service, and his treatment regimen consists of intensive moisturization with topical emollients, including Cetaphil® and a therapeutic shower oil (See Figure 3 for diagnostic timeline).

The third patient (P3), was born preterm at 30 weeks of gestation, whose clinical picture consisted of dilated cardiomyopathy, ventricular septal defect, incomplete atrioventricular canal, left pulmonary agenesis, macroglossia with glossoptosis, and cavum pellucidum septum. At birth, individual WES was performed and a heterozygous variant was identified, probably pathogenic in the GDF1 gene (c.885C>A; p.Tyr295*), associated with multiple types of congenital heart disease (MIM#613854). The patient had a fatal outcome at two days of age (See Figure 4 for diagnostic timeline).

Finally, patient 4 (P4), born on the day the test was requested, was referred due to respiratory distress at birth and a history of maternal death during childbirth due to nonmolecularly confirmed Marfan syndrome. It was inferred that the mother shared the FBN1 variant c.7704_7705insTGTG (p.Asp2569Cysfs*5) with her daughter, in whom it was identified and reported as likely pathogenic. The patient was clinically diagnosed with Marfan Syndrome (MIM#154700). Following the diagnosis, the patient was placed under the care of the cardiology service for periodic follow-up (See Figure 5 for diagnostic timeline).

Furthermore, we would like to emphasize a particular case, as it is the first patient affected with this condition in a cohort of more than 10,000 patients of all ages from the Specialized Laboratory of Clínica Colsanitas. He was a 78-day-old male patient with very low birth weight, severe respiratory symptoms, hyperlactatemia, profound hypoacusis in the right ear and severe hypoacusis in the left ear. He also had a prenatal history of severe intrauterine growth restriction (IUGR) and cytomegalovirus infection. In this patient, the homozygous variant TRMT10C c.542G>T (p.Arg181Leu) was identified, which is associated with combined oxidative phosphorylation deficiency 30, an ultra-rare systemic mitochondrial disease. Metodiev et al. (18) suggested through functional studies that this variant affects MRPP1 protein stability and mtRNA processing, without affecting m1R9 methyltransferase activity. Interestingly, the aCGH results in this patient identified loss of heterozygosity (LOH) greater than 5 Mb in 6 regions, including the 3q11.2q12.3 loci, where the TRMT10C gene is located.

Among the most frequently reported genes in exome sequencing, TPO was identified in two patients with compound heterozygosity, both presenting with hypothyroidism as their primary clinical concern. Pathogenic variants in TPO cause a severe form of congenital hypothyroidism, characterized by the immediate release of accumulated radioiodide following sodium perchlorate administration (OMIM #606765) (19). The European Society for Paediatric Endocrinology recommends genetic investigation of syndromic congenital hypothyroidism to identify novel genes and facilitate genetic counseling (20). In such cases, trio-based WES proves to be a valuable diagnostic tool, enabling early intervention and informed genetic counseling.

On the other hand, of the 125 aCGH tests performed, 103 were negative (no clinically significant CNV were identified), 10 were positive, and 12 revealed a CNV classified as VUS. In addition, among the cases without CNVs, 25 had LOH greater than 5 Mb, and in 4 of these cases WES was requested due to suspicion of a recessive hereditary disorder. The diagnostic yield of aCGH was 8%. Among the reported CNVs, 67% (n = 19) were duplications, while 32% (n = 9) were deletions. CNVs affected almost all chromosomes (1, 4, 5, 7, 8, 9, 11, 13, 15, 17, 20, 21, 22, X, and Y), with a higher frequency for chromosomes 17, X, and 21, as expected. Two of the patients who were detected CNV by WES underwent aCGH to confirm the finding.

One of the most frequent CNVs identified was chromosome 21 duplications (n = 3), which are associated with Down syndrome, included in the most common chromosomal aneuploidies (21). Affected patients presented with skeletal malformations, facial dysmorphism, and growth disorders. Trisomy 21 was clinically suspected in only one case, while the other two had not yet developed the characteristic phenotype, making diagnosis more challenging. Among these cases, two exhibited a partial duplication affecting only the q arm, while the third had a complete duplication of chromosome 21.

Furthermore, aCGH facilitated the identification of three patients exhibiting concurrent chromosomal loss and gain. The first case involved a five-day-old infant whose prenatal karyotyping at 22 weeks of gestation, conducted due to ultrasound findings of type III cleft palate and intrauterine growth restriction (IUGR), yielded normal results. Nevertheless, aCGH analysis revealed a 17.483 Mb gain on chromosome 4q34.1q35.2 and a 20.839 Mb loss on chromosome 7q34q36.3 (Figure 6), suggesting a balanced chromosomal rearrangement inherited from the father, as maternal karyotyping was normal. Notably, a similar chromosomal profile was reported in 2018, associated with single ventricle anomalies, partial thalamic fusion, and polycystic kidneys (22).

The second patient presented a loss of 15.4 Mb on 9p24.3p22.3 and a gain of 25.3 Mb on chromosome 11q22.3q25. Duplications in the 11q22.3q25 region have been reported in the literature in the context of translocations with other regions, associated with variable phenotypes such as dysmorphic facial features, minor cardiac anomalies, central nervous system anomalies and psychomotor retardation. For instance, Lekszas et al. (23), reported such an alteration by an unbalanced segregation of a paternal t(9;11)(p24.3;p15.4) translocation.

Finally, we identified a patient with a loss of 9.25 Mb on 1p36.33p36.22 and a gain of 31.5 Mb on 1q41q44. To our knowledge, no similar case reports have been published to date. The patient had prenatal findings of total agenesis of the corpus callosum, impaired neuronal migration and colpocephaly. The patient died three days after birth.

Among the 22 patients with CNV classified as pathogenic or VUS, clinical indications included: Multiple congenital malformations (n = 2), Genitourinary malformations (n = 2), Facial dysmorphism (n = 5), Growth disorders (n = 3), and Neurological disorder (n = 3). In particular, patients with facial dysmorphia had the highest number of reported pathogenic CNVs.