DYNC2H1 splicing variants causing severe prenatal short-rib polydactyly syndrome and postnatal orofaciodigital syndrome.

The DYNC2H1 gene has been associated with short-rib polydactyly syndrome (SRPS), among other skeletal ciliopathies. Two cases are presented of distinctive phenotypes resulting from splicing variants in DYNC2H1. The first is a 14-week-old fetus with enlarged nuchal translucency, oral hamartoma, malformed uvula, bifid epiglottis, short ribs, micromelia, long bone agenesis, polysyndactyly, heart defect, pancreatic cysts, multicystic dysplastic kidney, megabladder and trident acetabulum. A ciliopathies NGS panel revealed two compound heterozygous variants in DYNC2H1: c.7840-18T>G r.7841_7964del p.Gly2614Aspfs*5 and c.11070G>A r.11044_11116del p.Ile3682Aspfs*2. Both variants were initially classified as variants of uncertain significance but were reclassified as likely pathogenic after PCR-based RNA testing. The second is an 11-year-old overweight male with multiple accessory oral frenula, median cleft lip and alveolar ridge, polysyndactyly, brachydactyly, normal rib length, and hypogonadism. Exome sequencing revealed two compound heterozygous variants in DYNC2H1: c.6315del p.(Thr2106Glnfs*7), classified as likely pathogenic, and c.3303-16A>G p.(?), classified as a variant of uncertain significance. PCR-based RNA testing suggested that c.3303-16A>G induces an in-frame deletion: r.3303_3458del p.Asp1102_Arg1153del, although the normal transcript is still produced. These results are consistent with both SRPS type I/III in the first case and orofaciodigital syndrome in the second, an unprecedented description. This work thus improves the clinical and molecular knowledge of the phenotypes associated with splicing variants in the DYNC2H1 gene.

Familial juvenile hyperuricemic nephropathy: Revisiting the SLC8A1 gene, in a family with a novel terminal gross deletion in the UMOD gene.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) comprises a heterogeneous group of rare hereditary kidney diseases characterized by family history of progressive chronic kidney disease (CKD) with bland urine sediment, absence of significant proteinuria and normal or small-sized kidneys. Current diagnostic criteria require identification of a pathogenic variant in one of five genes - UMOD, MUC1, REN, HNF1ß, SEC61A1. The most prevalent form of ADTKD is uromodulin-associated kidney disease (ADTKD-UMOD). Genetic study of a Portuguese family diagnosed with familial juvenile hyperuricemic nephropathy (FJHN), one of the nosological entities in the spectrum of ADTKD, revealed a previously unreported large deletion in UMOD encompassing the entire terminal exon, which strictly cosegregated with CKD and hyperuricemia/gout, establishing the primary diagnosis of ADTKD-UMOD; as well as an ultra-rare nonsense SLC8A1 variant cosegregating with the UMOD deletion in patients that consistently exhibited an earlier onset of clinical manifestations. Since the terminal exon of UMOD does not encode for any of the critical structural domains or amino acid residues of mature uromodulin, the molecular mechanisms underlying the pathogenicity of its deletion are unclear and require further research. The association of the SLC8A1 locus with FJHN was first indicated by the results of a genome-wide linkage analysis in several multiplex families, but those data have not been subsequently confirmed. Our findings in this family revive that hypothesis.

A Novel Homozygous Splice Site Variant in AIMP1 Gene Causing Hypomyelinating Leukodystrophy: Case Report and Review of the Literature.

BACKGROUND: Biallelic pathogenic variants in AIMP1 gene cause hypomyelinating leukodystrophy type 3, a severe neurodegenerative disorder with early onset characterized by microcephaly, axial hypotonia, epilepsy, spasticity, and developmental delay. METHODS: Clinical exome sequence was performed on patient's DNA and Sanger sequencing was used to confirm the candidate variant. To better characterize the effect of the genetic variant, functional analysis based on Sanger sequencing of the proband's complementary DNA (cDNA) was performed. RESULTS: We report a case of 2-year-old girl with microcephaly, significant global developmental delay, refractory epilepsy, flaccid paralysis, hypomyelination, leukodystrophy, and cerebral atrophy on brain magnetic resonance imaging (MRI). Clinical exome sequencing revealed a novel splice site variant c.603 + 1G > A in homozygosity in the AIMP1 gene. Studies on patient's cDNA showed that the variant disrupts the canonical donor splice site of intron 5, with the recognition of a cryptic splice site within exon 5, leading to the skipping of the last 24 nucleotides of this exon together with the flanking intron. This alteration is predicted to cause an in-frame deletion of eight amino acids (p.Val194_Gln201del) belonging to the tRNA-biding domain of the protein. CONCLUSION: To the best of our knowledge, this is the first report of a splice site variant in the AIMP1 gene causing hypomyelinating leukodystrophy. The description of this patient not only expands the mutational spectrum of AIMP1 but also provides deeper insights on genotype-phenotype correlation by comparing the clinical features of our patient with previously reported affected individuals.

Whole-Exome Sequencing Targeting a Gene Panel for Sensorineural Hearing Loss: The First Portuguese Cohort Study.

Next-generation sequencing (NGS) technologies revolutionized the molecular diagnosis of sensorineural hearing loss (SNHL) and are now a standard of care. In this study, 71 Portuguese probands with hereditary SNHL were assessed by whole-exome sequencing (WES) targeting a panel of 158 genes related to SNHL, aiming to evaluate the diagnostic yield of this methodological approach and to report the spectrum of variants. Patients with either nonsyndromic or syndromic SNHL were included. Also, patients were previously screened for variants in the GJB2 gene and for duplications/deletions in the GJB6 gene. Causative variants in 11 different genes were identified in 15 (21.1%) out of 71 probands, 5 of which had associated syndromes. In 6 other patients (8.5%), presumptive causative variants were identified in MYO15A, TMIE, TBC1D24, SPMX, GJB3, PCDH15, and CDH23 genes, uncovering a potential case of digenic Usher syndrome. The study was inconclusive in 20 probands (28.2%), in 19 due to lack of segregation analysis and in one due to uncertain phenotype-genotype matching. In the remaining 30 patients (42.3%) no potentially causative variants were identified. The diagnostic yield did not significantly vary according to the age of hearing-impairment onset. As the first study on the application of NGS technologies in SNHL based on a Portuguese cohort, our results may contribute to characterize the spectrum of variants related to SNHL in the Portuguese population. Additionally, the present study provides new insights into the contribution of MYO3A, TECTA, EDNRB, TBC1D24, and GJB3 genes to SNHL. For the significant number of undiagnosed patients, reanalysis of WES data - either for a broader gene panel or in a non-targeted approach - may be considered.

The mutational spectrum of WT1 in male infertility.

PURPOSE: We evaluated the impact of WT1 mutations in isolated severe spermatogenic impairment in a population of European ancestry. WT1 was first identified as the gene responsible for Wilms tumor. It was later associated with a plethora of clinical phenotypes often accompanied by urogenital defects and male infertility. The recent finding of WT1 missense mutations in Chinese azoospermic males without major gonadal malformations broadened the phenotypic spectrum of WT1 defects and motivated this study. MATERIALS AND METHODS: We analyzed the WT1 coding region in a cohort of 194 Portuguese patients with nonobstructive azoospermia and in 188 with severe oligozoospermia with increased depth for the exons encoding the regulatory region of the protein. We also analyzed a group of 31 infertile males with a clinical history of unilateral or bilateral cryptorchidism and 1 patient with anorchia. RESULTS: We found 2 WT1 missense substitutions at higher frequency in patients than in controls. 1) A novel variant in exon 1 (p.Pro130Leu) that disrupted a mammalian specific polyproline stretch in the self-association domain was more frequent in azoospermia cases (0.27% vs 0.13%, p = 0.549). 2) A rare variant in a conserved residue in close proximity to the first zinc finger (pCys350Arg) was more frequent in severe oligozoospermia cases (0.80% vs 0.13%, p = 0.113). CONCLUSIONS: Results suggest a role for rare WT1 damaging variants in severe spermatogenic failure in populations of European ancestry. Large multicenter studies are needed to fully assess the contribution of WT1 genetic alterations to male infertility in the absence of other disease phenotypes.

A novel Alu-mediated microdeletion at 11p13 removes WT1 in a patient with cryptorchidism and azoospermia.

This article describes a patient with cryptorchidism and nonobstructive azoospermia presenting a novel microdeletion of approximately 1 Mb at 11p13. It was confirmed by multiplex ligation-dependent probe amplification that this heterozygous deletion spanned nine genes (WT1, EIF3M, CCDC73, PRRG4, QSER1, DEPDC7, TCP11L1, CSTF3 and HIPK3) and positioned the breakpoints within highly homologous repetitive elements. As far as is known, this is the smallest deletion as-yet described encompassing the WT1 gene and was detected only once in a total of 32 Portuguese patients with isolated uni- or bilateral cryptorchidism. These findings suggest that molecular analysis in patients with genitourinary features suggestive of WT1 impairment, namely cryptorchidism and renal abnormalities, may reveal cryptic genetic defects.

Alu-Alu Recombination Underlying the First Large Genomic Deletion in GlcNAc-Phosphotransferase Alpha/Beta (GNPTAB) Gene in a MLII Alpha/Beta Patient.

Mucolipidosis type II α/ß is a severe, autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/ß subunits of the GlcNAc-phosphotransferase. To date, over 100 different mutations have been identified in MLII α/ß patients, but no large deletions have been reported. Here we present the first case of a large homozygous intragenic GNPTAB gene deletion (c.3435-386_3602 + 343del897) encompassing exon 19, identified in a ML II α/ß patient. Long-range PCR and sequencing methodologies were used to refine the characterization of this rearrangement, leading to the identification of a 21 bp repetitive motif in introns 18 and 19. Further analysis revealed that both the 5' and 3' breakpoints were located within highly homologous Alu elements (Alu-Sz in intron 18 and Alu-Sq2, in intron 19), suggesting that this deletion has probably resulted from Alu-Alu unequal homologous recombination. RT-PCR methods were used to further evaluate the consequences of the alteration for the processing of the mutant pre mRNA GNPTAB, revealing the production of three abnormal transcripts: one without exon 19 (p.Lys1146_Trp1201del); another with an additional loss of exon 20 (p.Arg1145Serfs*2), and a third in which exon 19 was substituted by a pseudoexon inclusion consisting of a 62 bp fragment from intron 18 (p.Arg1145Serfs*16). Interestingly, this 62 bp fragment corresponds to the Alu-Sz element integrated in intron 18.This represents the first description of a large deletion identified in the GNPTAB gene and contributes to enrich the knowledge on the molecular mechanisms underlying causative mutations in ML II.

Incidence of maple syrup urine disease in Portugal.

Maple syrup urine disease is an autosomal recessive disorder of branched-chain amino acids metabolism with a worldwide frequency of 1/185,000 live newborns. In Portugal, the incidence of the disease has not been assessed. Based on the review of the cases diagnosed by tandem mass spectrometry an incidence of 1/86,800 live newborns was estimated in Portugal, indicating that the disease is more frequent in this country than reported in most populations.

Revisiting MSUD in Portuguese Gypsies: evidence for a founder mutation and for a mutational hotspot within the BCKDHA gene.

Maple syrup urine disease (MSUD) is a rare autosomal recessive disorder of branched-chain amino acid metabolism. In the context of the wide mutational spectrum known for this disease, a few common mutations have been described in populations where founder effects played a major role in modeling diversities. In Portugal, for instance, a high proportion of patients are of Gypsy origin and all share the same mutation (c.117delC-alpha; p.R40GfsX23), causing the neonatal severe form of MSUD. In this study, we used four microsatellite markers closely flanking the BCKDHA gene (E1alpha protein) to demonstrate that c.117delC-alpha is a founder mutation responsible for the high incidence of the disorder among Portuguese Gypsies. These results are of medical relevance since carrier tests and prenatal diagnosis can be offered to families at risk, particularly because the carrier frequency of c.117delC-alpha was estimated at 1.4% among the healthy Portuguese Gypsies from the South of the country. Finally we present evidence that the genomic region of the BCKDHA gene where c.117delC-alpha is located is likely a mutational hotspot, since recurrence of c.117delC-alpha was observed in two distinct population groups.

Molecular and structural analyses of maple syrup urine disease and identification of a founder mutation in a Portuguese Gypsy community.

Maple syrup urine disease (MSUD) is an autosomal recessive disorder, caused by the defective function of the branched-chain alpha-ketoacid dehydrogenase complex (BCKD). BCKD is a mitochondrial complex, encoded by four nuclear genes (BCKDHA, BCKDHB, DBT and DLD), involved in the metabolism of branched-chain amino acids (BCAAs). Since the MSUD mutational spectrum has not been previously assessed in Portugal, in this study we present the molecular characterization of 30 MSUD Portuguese patients. Seventeen putative mutations have been identified (six in BCKDHA, five in BCKDHB and six in DBT); seven of them are here described for the first time. The most common mutation identified was a C deletion in BCKDHA gene (c.117delC; p.R40GfsX23), already reported in the Spanish population. Interestingly, it was found in all patients of a Gypsy community from South of the country, so a founder effect is probably responsible for the high incidence of the disease in this community. Structural models of MSUD missense mutations have been performed to understand their pathogenic effect, in order to elucidate and often to predict the severity of a mutation clinical consequence.

The MTHFR C677T and A1298C polymorphisms and susceptibility to childhood acute lymphoblastic leukemia in Portugal.

Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in folate metabolism and in DNA methylation and synthesis. The role of two common polymorphisms at the MTHFR gene, C677T and A1298C, in the etiology of childhood or adult acute lymphoblastic leukemia (ALL) has been previously investigated. Although a protective effect of MTHFR*677T against ALL was systematically reported, the magnitude of the effect appeared to be influenced by population-specific gene-environmental interactions. The evidence of the role of MTHFR*1298C in ALL susceptibility was less consistent, emphasizing the need for enlarging molecular epidemiologic studies to independent trials from different populations. The authors analyzed in North Portugal the association between variations at the two MTHFR positions and risk of ALL by comparing genotypes and gene frequencies in 103 affected children with those in 111 healthy controls. None of the variations was found to significantly affect the risk of developing childhood ALL in North Portugal, and this finding per se is of relevance in further studies aimed at assessing the etiology of the pathology in this specific population. Despite the absence of statistical significance, these data revealed that the frequency of MTHFR*677T was lower in patients than in controls, a result that is congruent with other reports and with the functional model usually invoked to explain its ALL protective effect. Concerning MTHFR1298*C, this study failed to corroborate previous findings of decreased risk of ALL in the presence of the variant.