Untreated PKU patients without intellectual disability: SHANK gene family as a candidate modifier.

Phenylketonuria (PKU) is an inborn error of metabolism caused by variants in the phenylalanine hydroxylase (PAH) gene and it is characterized by excessively high levels of phenylalanine in body fluids. PKU is a paradigm for a genetic disease that can be treated and majority of developed countries have a population-based newborn screening. Thus, the combination of early diagnosis and immediate initiation of treatment has resulted in normal intelligence for treated PKU patients. Although PKU is a monogenic disease, decades of research and clinical practice have shown that the correlation between the genotype and corresponding phenotype is not simple at all. Attempts have been made to discover modifier genes for PKU cognitive phenotype but without any success so far. We conducted whole genome sequencing of 4 subjects from unrelated non-consanguineous families who presented with pathogenic mutations in the PAH gene, high blood phenylalanine concentrations and near-normal cognitive development despite no treatment. We used cross sample analysis to select genes common for more than one patient. Thus, the SHANK gene family emerged as the only relevant gene family with variants detected in 3 of 4 analyzed patients. We detected two novel variants, p.Pro1591Ala in SHANK1 and p.Asp18Asn in SHANK2, as well as SHANK2:p.Gly46Ser, SHANK2:p.Pro1388_Phe1389insLeuPro and SHANK3:p.Pro1716Thr variants that were previously described. Computational analysis indicated that the identified variants do not abolish the function of SHANK proteins. However, changes in posttranslational modifications of SHANK proteins could influence functioning of the glutamatergic synapses, cytoskeleton regulation and contribute to maintaining optimal synaptic density and number of dendritic spines. Our findings are linking SHANK gene family and brain plasticity in PKU for the first time. We hypothesize that variant SHANK proteins maintain optimal synaptic density and number of dendritic spines under high concentrations of phenylalanine and could have protective modifying effect on cognitive development of PKU patients.

Genetic characterization of GSD I in Serbian population revealed unexpectedly high incidence of GSD Ib and 3 novel SLC37A4 variants.

Glycogen storage disease (GSD) type I is inborn metabolic disease characterized by accumulation of glycogen in multiple organs. We analyzed 38 patients with clinical suspicion of GSD I using Sanger and next-generation sequencing (NGS). We identified 28 GSD Ib and 5 GSD Ia patients. In 5 patients, GSD III, VI, IX, cholesteryl-ester storage disease and Shwachman-Diamond syndrome diagnoses were set using NGS. Incidences for GSD Ia and GSD Ib were estimated at 1:172 746 and 1:60 461 live-births, respectively. Two variants were identified in G6PC gene: c.247C>T (p.Arg83Cys) and c.518T>C (p.Leu173Pro). In SLC37A4 gene, 6 variants were detected. Three previously reported variants c.81T>A (p.Asn27Lys), c.162C>A (p.Ser54Arg) and c.1042_1043delCT (p.Leu348Valfs*53) accounted for 87% of all analyzed alleles. Computational, transcription studies and/or clinical presentation in patients confirmed pathogenic effect of 3 novel variants: c.248G>A (p.Gly83Glu), c.404G>A (p.Gly135Asp) and c.785G>A (p.Ser263Glyfs*33 or p.Gly262Asp). In the cohort, hepatomegaly, hypoglycemia and failure to thrive were the most frequent presenting signs of GSD Ia, while hepatomegaly and recurrent bacterial infections were clinical hallmarks of GSD Ib. All GSD Ib patients developed neutropenia while 20.6% developed inflammatory bowel disease. Our study revealed the highest worldwide incidence of GSD Ib. Furthermore, description of 3 novel variants will facilitate medical genetic practice.

Genotype-phenotype correlation in 44 Czech, Slovak, Croatian and Serbian patients with mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome, MPS II, OMIM 309900) is an X-linked lysosomal storage disorder caused by deficiency of iduronate-2-sulfatase (IDS). We analyzed clinical and laboratory data from 44 Slavic patients with this disease. In total, 21 Czech, 7 Slovak, 9 Croatian and 7 Serbian patients (43 M/1 F) were included in the study (median age 11.0 years, range 1.2-43 years). Birth prevalence ranged from 1:69,223 (Serbia) to 1:192,626 (Czech Rep.). In the majority of patients (71%), the disease manifested in infancy. Cognitive functions were normal in 10 patients. Four, six and 24 patients had mild, moderate, and severe developmental delay, respectively, typically subsequent to developmental regression (59%). Residual enzyme activity showed no predictive value, and estimation of glycosaminoglycans (GAGs) had only limited importance for prognosis. Mutation analysis performed in 36 families led to the identification of 12 novel mutations, eight of which were small deletions/insertions. Large deletions/rearrangements and all but one small deletion/insertion led to a severe phenotype. This genotype-phenotype correlation was also identified in six cases with recurrent missense mutations. Based on patient genotype, the severity of the disease may be predicted with high probability in approximately half of MPS II patients.

Molecular and phenotypic characteristics of seven novel mutations causing branched-chain organic acidurias.

Specific mitochondrial enzymatic deficiencies in the catabolism of branched-chain amino acids cause methylmalonic aciduria (MMA), propionic acidemia (PA) and maple syrup urine disease (MSUD). Disease-causing mutations were identified in nine unrelated branched-chain organic acidurias (BCOA) patients. We detected eight previously described mutations: p.Asn219Tyr, p.Arg369His p.Val553Glyfs*17 in MUT, p.Thr198Serfs*6 in MMAA, p.Ile144_Leu181del in PCCB, p.Gly288Valfs*11, p.Tyr438Asn in BCKDHA and p.Ala137Val in BCKDHB gene. Interestingly, we identified seven novel genetic variants: p.Leu549Pro, p.Glu564*, p.Leu641Pro in MUT, p.Tyr206Cys in PCCB, p.His194Arg, p.Val298Met in BCKDHA and p.Glu286_Met290del in BCKDHB gene. In silico and/or eukaryotic expression studies confirmed pathogenic effect of all novel genetic variants. Aberrant enzymes p.Leu549Pro MUT, p.Leu641Pro MUT and p.Tyr206Cys PCCB did not show residual activity in activity assays. In addition, activity of MUT enzymes was not rescued in the presence of vitamin B12 precursor in vitro which was in accordance with non-responsiveness or partial responsiveness of patients to vitamin B12 therapy. Our study brings the first molecular genetic data and detailed phenotypic characteristics for MMA, PA and MSUD patients for Serbia and the whole South-Eastern European region. Therefore, our study contributes to the better understanding of molecular landscape of BCOA in Europe and to general knowledge on genotype-phenotype correlation for these rare diseases.