A Comprehensive Genetic Analysis of Slovenian Families with Multiple Cases of Orofacial Clefts Reveals Novel Variants in the Genes IRF6, GRHL3, and TBX22.

Although the aetiology of non-syndromic orofacial clefts (nsOFCs) is usually multifactorial, syndromic OFCs (syOFCs) are often caused by single mutations in known genes. Some syndromes, e.g., Van der Woude syndrome (VWS1; VWS2) and X-linked cleft palate with or without ankyloglossia (CPX), show only minor clinical signs in addition to OFC and are sometimes difficult to differentiate from nsOFCs. We recruited 34 Slovenian multi-case families with apparent nsOFCs (isolated OFCs or OFCs with minor additional facial signs). First, we examined IRF6, GRHL3, and TBX22 by Sanger or whole exome sequencing to identify VWS and CPX families. Next, we examined 72 additional nsOFC genes in the remaining families. Variant validation and co-segregation analysis were performed for each identified variant using Sanger sequencing, real-time quantitative PCR and microarray-based comparative genomic hybridization. We identified six disease-causing variants (three novel) in IRF6, GRHL3, and TBX22 in 21% of families with apparent nsOFCs, suggesting that our sequencing approach is useful for distinguishing syOFCs from nsOFCs. The novel variants, a frameshift variant in exon 7 of IRF6, a splice-altering variant in GRHL3, and a deletion of the coding exons of TBX22, indicate VWS1, VWS2, and CPX, respectively. We also identified five rare variants in nsOFC genes in families without VWS or CPX, but they could not be conclusively linked to nsOFC.

Early Discovery of Children With Lysosomal Acid Lipase Deficiency With the Universal Familial Hypercholesterolemia Screening Program.

Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive lysosomal storage disorder, caused by homozygous or compound heterozygous pathogenic variants in the LIPA gene. Clinically, LAL-D is under- and misdiagnosed, due to similar clinical and laboratory findings with other cholesterol or liver misfunctions. As a part of the Slovenian universal familial hypercholesterolemia (FH) screening, LAL-D is screened as a secondary condition among other rare dyslipidemias manifesting with hypercholesterolemia. Out of 669 children included, three were positive for a homozygous disease-causing splicing variant NM_000235.4: c.894G > A (NP_000226.2:p. Gln298Gln) in the LIPA gene (NG_008194.1). The mean age by the diagnosis of LAL-D was 9.8 ± 0.9 years. Moreover, all three LAL-D-positive children had an important elevation of transaminases and decreased activity of the lysosomal acid lipase enzyme. Abdominal MRI in all children detected an enlarged liver but a normal-sized spleen. In conclusion, universal FH screening algorithms with the confirmatory genetic analysis in the pediatric population enable also rare dyslipidemia detection at an early age. An important clinical criterion for differentiation between FH and the LAL-D-positive children has elevated transaminase levels (AST and ALT). In all three LAL-D positive children, an improvement in cholesterol and transaminase levels and steatosis of the liver has been seen after early treatment initiation.

Heterozygous NPR2 Variants in Idiopathic Short Stature.

Heterozygous variants in the NPR2 gene, which encodes the B-type natriuretic peptide receptor (NPR-B), a regulator of skeletal growth, were reported in 2-6% cases of idiopathic short stature (ISS). Using next-generation sequencing (NGS), we aimed to assess the frequency of NPR2 variants in our study cohort consisting of 150 children and adolescents with ISS, describe the NPR2 phenotypic spectrum with a growth pattern including birth data, and study the response to growth hormone (GH) treatment. A total of ten heterozygous pathogenic/likely pathogenic NPR2 variants and two heterozygous NPR2 variants of uncertain significance were detected in twelve participants (frequency of causal variants: 10/150, 6.7%). During follow-up, the NPR2 individuals presented with a growth pattern varying from low-normal to significant short stature. A clinically relevant increase in BMI (a mean gain in the BMI SDS of +1.41), a characteristic previously not reported in NPR2 individuals, was observed. In total, 8.8% participants born small for their gestational age (SGA) carried the NPR2 causal variant. The response to GH treatment was variable (SDS height gain ranging from -0.01 to +0.74). According to the results, NPR2 variants present a frequent cause of ISS and familial short stature. Phenotyping variability in growth patterns and variable responses to GH treatment should be considered.

An Adolescent Boy with Klinefelter Syndrome and 47,XXY/46,XX Mosaicism: Case Report and Review of Literature.

Klinefelter syndrome is the most commonly reported sex chromosome abnormality. It is heavily underdiagnosed due to the substantial variability of clinical presentations but is generally characterized by small, firm testes, hypergonadotropic hypogonadism, and the absence of spermatogenesis. Most patients with Klinefelter syndrome have a 47,XXY genotype. If they present with mosaicism, two different cell lines are usually identified, an aneuploid 47,XXY cell line and a normal male 46,XY cell line. There are very few cases of 47,XXY mosaicism with the additional female cell line 46,XX described in the literature. We report a case of an adolescent with the male phenotype and a rare variant mosaic 47,XXY/46,XX karyotype who presented with painless bilateral gynaecomastia. 47,XXY and 46,XX mosaic cell lines were identified with GTG-banding and further characterized using fluorescent in situ hybridization. We summarized the available clinical presentations of reported male patients with 47,XXY/46,XX mosaicism. To improve the clinical management and quality of life in individuals with rare and cryptic genomic imbalances, the genetic diagnosis would need to be extended to atypical cases.

Genetic Variability in Slovenian Cohort of Patients with Oculocutaneous Albinism.

Oculocutaneous albinism (OCA) is an inherited disorder affecting the visual system and skin pigmentation. Our aim was to evaluate genetic and clinical heterogeneity in a cohort of Slovenian paediatric patients with clinically suspected OCA using advanced molecular-genetics approach. In as much as 20 out of 25 patients, genetic variants explaining their clinical phenotype were identified. The great majority of patients (15/25) had genetic variants in TYR gene associated with OCA type 1, followed by variants in TYRP1, SLC45A2 and HPS1 genes causative for OCA3, OCA4 and Hermansky-Pudlak syndrome type 1, respectively. We concluded that OCA phenotype could not predict genotype and vice versa. Nevertheless, the diagnostic yield after targeted next generation sequencing (NGS) was 80% and proved to be affective in our paediatric cohort of patients with various degree of OCA. Even in 16 patients with normal complexion the diagnostic yield was 62,5%. Interestingly, we have identified a patient of white European ancestry with OCA3, which is an extremely rare report, and one patient with OCA due to the Hermansky-Pudlak syndrome type 1.

Detection of Del/Dup Inside SHOX/PAR1 Region in Children and Young Adults with Idiopathic Short Stature.

Short stature is a common growth disorder defined as a body height two standard deviations (SD) or more below the mean for a given age, gender, and population. A large part of the cases remains unexplained and is referred to as having idiopathic short stature (ISS). One of the leading genetic causes of short stature is variants of short stature homeobox-containing gene (SHOX) and is considered to be responsible for 2-15% of ISS. We aimed to analyse the regulatory and coding region of SHOX in Slovenian children and young adults with ISS and to investigate the pathogenicity of detected variants. Our cohort included 75 children and young adults with ISS. Multiplex ligation-dependent probe amplification (MLPA) was performed in all participants for the detection of larger copy number variations (CNVs). Sanger sequencing was undertaken for the detection of point variants, small deletions, and insertions. A total of one deletion and two duplications were discovered using the MLPA technique. Only one of these four variants was identified as disease-causing and occurred in one individual, which represents 1.3% of the cohort. With Sanger sequencing, two variants were discovered, but none of them appeared to have a pathogenic effect on height. According to the results, in the Slovenian population of children and young adults with ISS, SHOX deficiency is less frequent than expected considering existing data from other populations.

Dual Role of PTPN22 but Not NLRP3 Inflammasome Polymorphisms in Type 1 Diabetes and Celiac Disease in Children.

Genetic polymorphisms in genes coding for inflammasome components nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain-containing protein 3 (NLRP3) and caspase recruitment domain-containing protein 8 (CARD8) have been associated with autoinflammatory and autoimmune diseases. On the other hand several studies suggested that NLRP3 inflammasome contributes to maintenance of gastrointestinal immune homeostasis and that activation of NLRP3 is regulated by protein tyrosine phosphatase non-receptor 22 (PTPN22). PTPN22 polymorphism was implicated in the risk for various autoimmune diseases including type 1 diabetes (T1D) but not for celiac disease (CD). The aim of our study was to evaluate the role of inflammasome related polymorphisms in subjects with either T1D or CD as well as in subjects affected by both diseases. We examined PTPN22 rs2476601 (p.Arg620Trp), NLRP3 rs35829419 (p.Gln705Lys), and CARD8 rs2043211 (p.Cys10Ter) in 66 subjects with coexisting T1D and CD, 65 subjects with T1D who did not develop CD, 67 subjects diagnosed only with CD and 127 healthy unrelated Slovenian individuals. All results were adjusted for clinical characteristic and human leukocyte antigen (HLA) risk. PTPN22 rs2476601 allele was significantly more frequent among subjects with T1D (Padj = 0.001) and less frequent in subjects with CD (Padj = 0.039) when compared to controls. In patients with coexisting T1D and CD this variant was significantly less frequent compared to T1D group (Padj = 0.010). Protective effect on CD development in individuals with T1D was observed only within the low risk HLA group. On the other hand, we found no association of NLRP3 rs35829419 and CARD8 rs2043211 with the development of T1D, CD or both diseases together. In conclusion PTPN22 rs2476601polymorphism was significantly associated with the risk of developing T1D in Slovenian population, while no associations of proinflammatory NLRP3 and CARD8 polymorphisms with T1D and CD were observed. Interestingly, the same PTPN22 variant protected from CD. We hypothesize that this effect may be mediated through the NLRP3 inflammasome activation.

Metabolic control, ApoE genotypes, and dyslipidemia in children, adolescents and young adults with type 1 diabetes.

BACKGROUND AND AIMS: Limited data is available on the factors influencing the lipid profiles and the prevalence of dyslipidemia in children, adolescents and young adults with type 1 diabetes. We aimed at assessing the influences of metabolic control and ApoE genotypes on lipid profiles and the prevalence of dyslipidemia in children, adolescents and young adults with type 1 diabetes. METHODS: Children, adolescents and young adults with type 1 diabetes from our nationwide cohort attending the annual check-up were prospectively included. Data on metabolic control and expanded lipid profiles were collected, and ApoE genotyping performed. Test for homoscedasticity of continuous variables was followed by ANOVA and Welch's ANOVA tests, and Chi-square test was used for categorical variables with Kruskal-Wallis test as a control. RESULTS: 467 patients were included in the data analysis: 226 female (48.4%), mean age 14.71 ±â€¯5.09 years and diabetes duration 6.74 ±â€¯4.54 years. Mean HbA1c was 61 ±â€¯5 mmoL/mol (7.71 ±â€¯1.22%), with no gender-related differences. Females had higher mean total cholesterol (p < 0.001), LDL-C (p = 0.005), HDL-C (p < 0.001), non-HDL-C (p = 0.003), and ApoB levels (p < 0.001). 26.3% of participants had LDL-C levels above the type 1 diabetes LDL-C-goal of 2.6 mmoL/L, and 19.5% had elevated/borderline-elevated lipoprotein(a) (Lp(a)). HbA1c levels were positively related to higher levels of LDL-C (p = 0.0070) and Lp(a) (p = 0.0020). Participants with ApoE4(e3/e4) allele had higher levels of LDL-C (p = 0.010), independently of HbA1c. CONCLUSIONS: Females and subjects with suboptimal metabolic control had more adverse lipid profiles. ApoE4(e3/e4) alleles were associated with significantly higher LDL-C levels, independently of HbA1c.

Characterization of a de novo sSMC 17 detected in a girl with developmental delay and dysmorphic features.

BACKGROUND: The majority of small supernumerary marker chromosome cases arise de novo and their frequency in newborns is 0.04%. We report on a girl with developmental delay and dysmorphic features with a non-mosaic de novo sSMC that originated from the pericentric region of q arm in chromosome 17. CASE PRESENTATION: The girl presented with developmental delay, speech delay, myopia, mild muscle hypotonia, hypoplasia of orbicular muscle, poor concentration, and hyperactivity. Main dysmorphic features included: round face, microstomia, small chin, down-slanting palpebral fissures and small lobules of both ears. At present, her developmental abilities are still delayed for her chronological age but she is making evident progress with speech. A postnatal array comparative genomic hybridization showed a 2.31 Mb genomic gain indicating microduplication derived from pericentric regions q11.1 and q11.2 of chromosome 17. Additional conventional cytogenetic analysis from peripheral blood characterized the karyotype as 47,XX,+mar in a non-mosaic form. The location of microduplication was confirmed with fluorescence in situ hybridization. CONCLUSION: The proband's microduplication encompassed approximately 40 annotated genes, several of which have been associated with phenotypic characteristics of the proband. This is the first report of sSMC 17 including this particular chromosomal region in non-mosaic form.

11q terminal deletion and combined immunodeficiency (Jacobsen syndrome): Case report and literature review on immunodeficiency in Jacobsen syndrome.

Antibody deficiency is common finding in patients with Jacobsen syndrome (JS). In addition, there have been few reports of T-cell defects in this condition, possibly because most of the reported patients have not been specifically evaluated for T-cell function. In this article, we present a child with an 11q deletion and combined immunodeficiency and we perform a literature overview on immunodeficiency in JS. Our patient presented with recurrent bacterial and prolonged viral infections involving the respiratory system, as well as other classic features of the syndrome. In addition to low IgM, IgG4, and B-cells, also low recent thymic emigrants, helper and naïve T-cells were found. We propose that patients with Jacobsen syndrome need thorough immunological evaluations as T-cell dysfunction might be more prevalent than previously reported. Patients with infections consistent with T-cell defects should be classified as having combined immunodeficiency. © 2016 Wiley Periodicals, Inc.

Neonatal Cyanosis Due to Hemoglobin Variant: Hb F-Sarajevo.

Neonatal cyanosis is rarely due to hemoglobin variants with low oxygen affinity. We describe the clinical course and results of molecular genetic analysis of a boy who presented after birth with severe cyanosis. Arterial blood-gas analysis demonstrated a pronounced shift of the oxygen-hemoglobin dissociation curve to the right and molecular genetic analysis revealed a γ-globin variant, Hb F-Sarajevo. The patient presented is the second reported case of neonatal cyanosis due to this mutation, which was first described in 2012 by Zimmermann-Baer and coauthors. With the introduction of universal screening for congenital heart disease, the finding of low oxygen saturation will uncover more neonates with hemoglobinopathies with low oxygen affinity.

Clinical Role of CYP2C19 Polymorphisms in Patients with Congenital Adrenal Hyperplasia Due to 21-hydroxylase Deficiency.

Extraadrenal enzymes such as CYP2C19 may participate in residual 21-hydroxylation of progesterone leading to milder phenotypes of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD). Among 94 21OHD patients 28 were homozygous or compound heterozygous for severe CYP21A2 mutations. We have reviewed their clinical phenotype and obtained information on maintenance doses of hydrocortisone and fludrocortisone. All patients were genotyped for CYP2C19*2 and CYP2C19*17 alleles. Eleven patients with CYP2C19*1/*17 genotype had all salt-wasting 21OHD. Among 17 patients with CYP2C19 genotypes leading to normal or decreased CYP2C19 activity, 15 had salt-wasting, one had simple virilizing and one had non-classical 21OHD. CYP2C19*1/*17 genotype was associated with lower maintenance dose of fludrocortisone (p = 0.04), but not of hydrocortisone (p > 0.05). Increased CYP2C19 activity could slightly ameliorate mineralocorticoid deficiency in 21OHD.

The association of folate pathway and DNA repair polymorphisms with susceptibility to childhood acute lymphoblastic leukemia.

Genetic factors may play an important role in susceptibility to childhood acute lymphoblastic leukemia (ALL). The aim of our study was to evaluate the associations of genetic polymorphisms in folate pathway and DNA repair genes with susceptibility to ALL. In total, 121 children with ALL and 184 unrelated healthy controls of Slovenian origin were genotyped for 14 polymorphisms in seven genes of folate pathway, base excision repair and homologous recombination repair (TYMS, MTHFR, OGG1, XRCC1, NBN, RAD51, and XRCC3). In addition, the exon 6 of NBN was screened for the presence of mutations using denaturing high performance liquid chromatography. Twelve polymorphisms were in Hardy-Weinberg equilibrium in controls and their genotype frequencies were in agreement with those reported in other Caucasian populations. Among the investigated polymorphisms and mutations, NBN Glu185Gln significantly decreased susceptibility to B-cell ALL (p=0.037), while TYMS 3R allele decreased susceptibility to T-cell ALL (p=0.011). Moreover, significantly decreased susceptibility to ALL was observed for MTHFR TA (p=0.030) and RAD51 GTT haplotypes (p=0.016). Susceptibility to ALL increased with the increasing number of risk alleles (ptrend=0.007). We also observed significant influence of hOGG-RAD51 and NBN-RAD51 interactions on susceptibility to ALL. Our results suggest that combination of several polymorphisms in DNA repair and folate pathways may significantly affect susceptibility to childhood ALL.

Clinical and Molecular Cytogenetic Characterisation of Children with Developmental Delay and Dysmorphic Features.

INTRODUCTION: Developmental delay and dysmorphic features affect 1 - 3 % of paediatric population. In the last few years molecular cytogenetic high resolution techniques (comparative genomic hybridization arrays and single-nucleotide polymorphism arrays) have been proven to be a first-tier choice for clinical diagnostics of developmental delay and dysmorphic features. METHODS AND RESULTS: In the present article we describe the clinical advantages of molecular cytogenetic approach (comparative genomic hybridization arrays and single nucleotide polymorphism arrays) in the diagnostic procedure of two children with developmental delay, dysmorphic features and additional morphological phenotypes. Additionally, we demonstrate the necessity of fluorescent in situ hybridization utilisation to identify the localisation and underlying mechanism of detected chromosomal rearrangement. CONCLUSIONS: Two types of chromosomal abnormalities were identified and confirmed using different molecular genetic approaches. Comparative genomic hybridization arrays and single nucleotide polymorphism arrays are hereby presented as important methods to identify chromosomal imbalances in patients with developmental delay and dysmorphic features. We emphasize the importance of molecular genetic testing in patients' parents for the demonstration of the origin and clinical importance of the aberrations prior determined in the patients. The results obtained using molecular cytogenetic high resolution techniques methods are the cornerstone for proper genetic counselling to the affected families.

Association of Average Telomere Length with Body-Mass Index and Vitamin D Status in Juvenile Population with Type 1 Diabetes.

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune chronic disease where hyperglycemia, increased risk of oxidative stress, advanced glycation end-products and other genetic and environmental factors lead to T1D complications. Shorter telomeres are associated with hyperglycemic levels and lower serum vitamin D levels. METHODS: Average telomere length (ATL) in whole blood DNA samples was assessed with qPCR method in 53 Slovenian T1D children/adolescents (median age 8.7 years, 1:1.3 male/female ratio). Body mass index standard deviation score (BMI-SDS), glycated haemoglobin and serum level of vitamin D metabolite (25-(OH)-D3) and the age at the onset of T1D were collected from the available medical documentation. RESULTS: Results indicate shorter ATL in subjects with higher BMI-SDS when compared to those with longer ATL (0.455 ± 0.438, -0.63 ± 0.295; p=0.049). Subjects with higher BMI-SDS had lower serum vitamin D levels when compared to those with lower BMI-SDS (40.66 ± 3.07 vs. 52.86 ± 4.85 nmol/L; p=0.045). Vitamin D serum levels did not significantly differ between subjects with longer/shorter ATL. CONCLUSION: T1D children/adolescents with shorter ATL tend to have higher BMI-SDS. Lower serum vitamin D levels were associated with higher BMI-SDS, while associations between vitamin D serum levels, age at the onset of T1D, glycated haemoglobin and ATL were not observed. Additional studies with more participants are required to clarify the role of the telomere dynamics in T1D aetiology and development of complications.

Cytogenetic and Molecular Genetic Characterization of Children with Short Stature.

BACKGROUND: The deficiency of SHOX gene (short stature homeobox-containing gene) has been recognized as the most frequent monogenetic cause of short stature. SHOX gene has been associated with short stature in Turner syndrome and Leri Weill dyschondrosteosis as well with non-syndromic idiopathic short stature. The aim of this study was to determine the frequency of SHOX deletions and mutations in a cohort of Slovenian children with short stature, and to delineate indications for routine SHOX gene mutation screening. METHODS AND RESULTS: 40 selected subjects with idiopathic short stature were screened for entire SHOX gene deletion and for mutations in the SHOX gene coding region (exon 2 to 6), together with sequences flanking the exon-intron boundaries. FISH analysis on metaphase and interphase spreads revealed no entire gene deletion. Additionally, no pathogenic point mutations or smaller deletion/duplications were identified in this study group. CONCLUSIONS: SHOX gene deletions and point mutations are not a common cause of idiopathic short stature in a cohort of Slovenian children with short stature. Therefore, the frequency of SHOX mutations must be much lower as expected based on the reported data.

Severe progressive obstructive cardiomyopathy and renal tubular dysfunction in Donohue syndrome with decreased insulin receptor autophosphorylation due to a novel INSR mutation.

UNLABELLED: Donohue syndrome (leprechaunism; OMIM *246200) is a rare, recessively inherited disorder of extreme insulin resistance due to mutations in the insulin receptor gene (INSR) causing either defects in insulin binding or receptor autophosphorylation and tyrosine kinase activity. We report a patient with pronounced clinical picture of leprechaunism who developed severe progressive hypertrophic obstructive cardiomyopathy (HOCM) and renal tubular dysfunction which improved on continuous subcutaneous infusion of recombinant human insulin-like growth factor-1 (rhIGF-I). INSR gene molecular analysis and insulin receptor (IR) autophosphorylation on cultured fibroblasts were performed. A novel homozygous missense mutation p.Leu795Pro was found, located in the extracellular portion of the ß subunit of the insulin receptor. The post-binding defect of the insulin receptor signaling in cultured fibroblasts demonstrated decreased insulin receptor autophosphorylation. CONCLUSION: Treatment with rhIGF-I partially reversed severe progressive HOCM and renal tubular dysfunction in a patient with Donohue syndrome associated with a novel p.Leu795Pro INSR gene mutation causing a severe decrease in IR autophosphorylation.

GPR143 gene mutation analysis in pediatric patients with albinism.

BACKGROUND: X-linked ocular albinism type 1 is difficult to differentiate clinically from other forms of albinism in young patients. X-linked ocular albinism type 1 is caused by mutations in the GPR143 gene, encoding melanosome specific G-protein coupled receptor. Patients typically present with moderately to severely reduced visual acuity, nystagmus, strabismus, photophobia, iris translucency, hypopigmentation of the retina, foveal hypoplasia and misrouting of optic nerve fibers at the chiasm. MATERIALS AND METHODS: Following clinical ophthalmological evaluation, GPR143 gene mutational analyses were performed in a cohort of 15 pediatric male patients with clinical signs of albinism. RESULTS: Three different mutations in the GPR143 gene were identified in four patients, including a novel c.886G>A (p.Gly296Arg) mutation occurring "de novo" and a novel intronic c.360 + 5G>A mutation, identified in two related boys. CONCLUSIONS: Four patients with X-linked ocular albinism type 1 were identified from a cohort of 15 boys with clinical signs of albinism using mutation detection methods. Genetic analysis offers the possibility of early definitive diagnosis of ocular albinism type 1 in a significant portion of boys with clinical signs of albinism.

Five novel mutations and two large deletions in a population analysis of the phenylalanine hydroxylase gene.

Mutational spectrum of the phenylalanine hydroxylase (PAH) deficiency was investigated in 107 families (90% of the Slovene PKU population). The entire coding region of the PAH gene was analyzed with dHPLC to select the samples where subsequently the automated sequencing analysis was performed. MLPA analysis was performed to identify large deletions, which were later confirmed with long-range PCR. Correlations with patients' phenotypes and genotype-based predictions of BH(4)-responsiveness were assessed. Altogether, disease-causing mutations were identified on 209 alleles (detection rate 97.7%). A spectrum of 36 different disease-causing mutations was identified: 20 missense mutations (80% of the alleles), eight splicing mutations (13% of the alleles), one nonsense mutation (0.5% of the alleles), four small deletions with frame shift (6% of the alleles), one small insertion with frame shift (0.5% of the alleles), and two large deletions (2% of the alleles). The most frequent mutation was p.R408W in exon 12, representing 29% of the alleles, which is in concordance with other neighboring and/or Slavic PKU populations. Other common mutations were: p.R158Q, p.A403V, p.P281L and p.E390G, accounting for 9%, 7%, 7% and 7% of the alleles respectively. Five novel mutations were detected: c.43_44insAG, c.56_59+1delACAGG, p.V45A, p.L62P and p.R157S. Large deletion of exon 5 (EX5del955) was found in three patients and a deletion of exon 3 (EX3del4765) in one patient. A spectrum of 64 different genotypes was found, seven of them accounting for over than a third of all families. Among thirteen families with homozygous mutation (13% of the PKU population), 10 had p.R408W, two had p.R158Q and one had p.E390G. Among 107 families, 58 were classified as classic PKU (54.2%), 28 as mild PKU (25.9%) and 21 as MHP (19.6%). Twenty-six different genotypes (40.6%) were predicted to be BH(4)-responsive, represented by 38 different families (35.5%).