Phenotype expansion and development in Kosaki overgrowth syndrome.

We expand the Kosaki overgrowth syndrome (KOGS) phenotype by over 70% to include 24 unreported KOGS symptoms, in a first male patient, the third overall associated with the PDGFRB c.1751C>G p.(Pro584Arg) mutation. Eighteen of these symptoms are unique to our patient, the remaining six are shared with other patients. Of the 24 unreported features overall, 6 show marked phenotype evolution and varying time of onset. The triangular face detected at 14 months and long palpebral fissures with lateral ectropion at 4 years are present in other members of the cohort. The remaining 4 are unique to Patient 5: pronounced macrocephaly from birth, increasingly triangular anterior skull from 14 months, camptodactyly, emerging at 4 years and worsening joint contractures from 6 years. Compilation of all new symptoms reported here with published clinical data further identifies at least 18 clinical parameters common to all cases to date, encompassing both known KOGS-associated PDGFRB mutations. We therefore propose a set of 18 core KOGS symptoms, with 16 present in early childhood. These results should also impact diagnostic/prognostic scope, intervention and outcome potential for KOGS patients, particularly for developmentally progressive conditions such as scoliosis and myofibroma.

PHENOTYPIC CONSEQUENCES AND THE MALIGNANCY RISK IN FAMILIAL NOONAN SYNDROME DUE TO A RARE P.S427G RAF1 MUTATION.

Mutations leading to dysregulation of the Ras/MAPK signal transduction cascade are a common cause of Noonan syndrome (NS) and play a key role in the pathogenesis of many human malignancies. To date, about 24 various RAF1 germline mutations were identified in NS. The incidence of malignancies in NS patients with RAF1 mutations has not been reported so far. However, in a few cases somatic RAF1 mutations were observed in cancer, including two described in therapy-related acute myeloid leukaemia (t-AML). We present a case of an adult female patient with Noonan syndrome and her affected mother with a rare RAF] germline mutation c.1279A>G (p.S427G), located within the highly conserved domain (CR3) of serine/threonine kinase C-RAF. Interestingly, this mutation has been reported for the first time in a patient with t-AML as a somatic change and so far has been identified in only one individual with NS phenotype and his mother. Our report presents the second familial case of Noonan syndrome due to a germline p.S427G substitution in RAF] with no occurrence of a malignant tumor. It may suggest that carrying a germline mutation in the RAF1 oncogene is not associated with an increased risk of tumor development. Since RAF1 mutations have been observed as a somatic event in many types of cancer, this report might be of importance for the genetic counselling and management of patients both with germline and somatic alterations in this gene.

MOLAR TOOTH SIGN AND ACROCALLOSAL SYNDROME--A REPORT ON A POLISH FAMILY AND REVIEW OF KIF7 SYNDROMOLOGY.

Acrocallosal syndrome is a multiple congenital anomaly disorder characterized by postaxial and/or preaxial polydactyly, cutaneous syndactyly, macrocephaly, widely spaced eyes, absence or hypoplasia of the corpus callosum, and intellectual disability. It was first described by Albert Schinzel as early as in 1979, but the diagnosis of this syndrome still remains challenging. Here we report a family with 2 sibs with acrocallosal syndrome caused by novel mutations in KIF7. They present with features like molar tooth sign and hyperventilation that are not very typical in ACLS, but do occur in other ciliopathies, hence we also discuss the clinical heterogeneity of KIF7-associated disorders.

Novel c.191C>G (p.Pro64Arg) MPV17 mutation identified in two pairs of unrelated Polish siblings with mitochondrial hepatoencephalopathy.

This study reports clinical, biochemical and histopathological findings associated with a novel homozygous MPV17 mutation in four patients with mitochondrial depletion syndrome. The severe course of the disease, which started in the first weeks of life, was dominated by a failure to thrive, hypotonia and liver dysfunction, with relatively mild neurological involvement. All affected infants died by 1 year of age. Laboratory findings included progressive liver failure (hypertransaminasaemia, icterus, and coagulopathy), recurrent hypoglycaemia, lactic acidaemia, hyperferritinaemia, and increased transferrin saturation. Histological and ultrastructural analyses uncovered significant lipid accumulation in hepatocytes and myocytes. A severe decrease in the mitochondrial/nuclear DNA (mtDNA/nDNA) ratio was found post-mortem in the livers (and in one muscle specimen) of both examined patients. Oxidative phosphorylation system (OXPHOS) Western blotting revealed low levels of complexes I, III and IV subunits. The highlights of our findings are as follows: (i) The novel p.Pro64Arg mutation is the second recurrent MPV17 mutation reported. The phenotype associated with the p.Pro64Arg mutation differs from the phenotype of the relatively common p.Arg50Gln mutation, suggesting the existence of a genotype-phenotype correlation. (ii) Tissues collected from patients during autopsy may be useful for both mtDNA/nDNA ratio assessment and OXPHOS Western blotting.

De novo deletions of SNRPN exon 1 in early human and mouse embryos result in a paternal to maternal imprint switch.

Prader-Willi syndrome (PWS) is a neurogenetic disease characterized by infantile hypotonia, gonadal hypoplasia, obsessive behaviour and neonatal feeding difficulties followed by hyperphagia, leading to profound obesity. PWS is due to a lack of paternal genetic information at 15q11-q13 (ref. 2). Five imprinted, paternally expressed genes map to the PWS region, MKRN3 (ref. 3), NDN (ref. 4), NDNL1 (ref. 5), SNRPN (refs 6-8 ) and IPW (ref. 9), as well as two poorly characterized framents designated PAR-1 and PAR-5 (ref. 10). Imprinting of this region involves a bipartite 'imprinting centre' (IC), which overlaps SNRPN (refs 10,11). Deletion of the SNRPN promoter/exon 1 region (the PWS IC element) appears to impair the establishment of the paternal imprint in the male germ line and leads to PWS. Here we report a PWS family in which the father is mosaic for an IC deletion on his paternal chromosome. The deletion chromosome has acquired a maternal methylation imprint in his somatic cells. We have made identical findings in chimaeric mice generated from two independent embryonic stem (ES) cell lines harbouring a similar deletion. Our studies demonstrate that the PWS IC element is not only required for the establishment of the paternal imprint, but also for its postzygotic maintenance.

Anthropometric characteristics of 65 Polish Smith-Lemli-Opitz patients.

Smith-Lemli-Opitz syndrome (SLOS) belongs to a group of multiple congenital anomaly/developmental delay disorders. Its primary cause lies in the defect in cholesterol biosynthesis-7-dehydrocholesterol reductase (DHCR7)-caused by pathogenic variants in the homonymous gene. Anthropometric anomalies, especially growth restriction and microcephaly, are among the most common physical manifestations of SLOS. There have been no studies analyzing the correlation between genotype, biochemical marker (7-dehydrocholesterol), and the birth and growth parameters for individuals with SLOS. This paper presents anthropometric data from the group of 65 Polish patients (aged 0.1 to 18 years) with Smith-Lemli-Opitz syndrome, with genotype and biochemical correlations for birth parameters, as well as growth in relation to molecular DHCR7 variants.

Epigenetic mutations of the imprinted IGF2-H19 domain in Silver-Russell syndrome (SRS): results from a large cohort of patients with SRS and SRS-like phenotypes.

BACKGROUND: Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous condition characterised by severe intrauterine and postnatal growth retardation. Loss of DNA methylation at the telomeric imprinting control region 1 (ICR1) on 11p15 is an important cause of SRS. METHODS: We studied the methylation pattern at the H19-IGF2 locus in 201 patients with suspected SRS. In an attempt to categorise the patients into different subgroups, we developed a simple clinical scoring system with respect to readily and unambiguously assessable clinical features. In a second step, the relationship between clinical score and epigenetic status was analysed. RESULTS AND CONCLUSIONS: The scoring system emerged as a powerful tool for identifying those patients with both a definite SRS phenotype and carrying an epimutation at 11p15. 53% of the 201 patients initially enrolled fulfilled the criteria for SRS and about 40% of them exhibited an epimutation at the H19-IGF2 locus. Methylation defects were restricted to patients who fulfilled the diagnostic criteria for SRS. Patients carrying epimutations had a more severe phenotype than either the SRS patients with mUPD7 or the idiopathic SRS patients. The majority of patients with methylation abnormalities showed hypomethylation at both the H19 and IGF2 genes. However, we also identified SRS patients where hypomethylation was restricted to either the H19 or the IGF2 gene. Interestingly, we detected epimutations in siblings of normal parents, most likely reflecting germ cell mosaicism in the fathers. In one family, we identified an epimutation in an affected father and his likewise affected daughter.

SURF1 missense mutations promote a mild Leigh phenotype.

UNLABELLED: SURF1 gene mutations are the most common cause of Leigh syndrome (LS), a rare progressive neurodegenerative disorder of infancy, characterized by symmetric necrotizing lesions and hypervascularity in the brainstem and basal ganglia, leading to death before the age of 4 years. Most of the reported mutations create premature termination codons, whereas missense mutations are rare. The aim of the study was to characterize the natural history of LS patients carrying at least one missense mutation in the SURF1 gene. Nineteen such patients (8 own cases and 11 reported in the literature) were compared with a reference group of 20 own c.845_846delCT homozygous patients, and with other LS(SURF-) cases described in the literature. Disease onset in the studied group was delayed. Acute failure to thrive and hyperventilation episodes were rare, respiratory failure did not appear before the age of 4 years. Dystonia, motor regression and eye movement dissociation developed slowly. The number of patients who survived 7 years of life totaled 9 out of 15 (60%) in the 'missense group' and 1 out of 26 (4%) patients with mutations leading to truncated proteins. IN CONCLUSION: (i) The presence of a missense mutation in the SURF1 gene may correlate with a milder course and longer survival of Leigh patients, (ii) normal magnetic resonance imaging (MRI) findings, normal blood lactate value, and only mild decrease of cytochrome c oxidase (COX) activity are not sufficient reasons to forego SURF1 mutation analysis in differential diagnosis.

Age and origin of major Smith-Lemli-Opitz syndrome (SLOS) mutations in European populations.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) (MIM 270 400) is an autosomal recessive multiple congenital anomalies/mental retardation syndrome caused by mutations in the Delta7-sterol reductase (DHCR7, E.C.1.3.1.21) gene. The prevalence of SLOS has been estimated to range between 1:15000 and 1:60000 in populations of European origin. METHODS AND RESULTS: We have analysed the frequency, origin, and age of DHCR7 mutations in European populations. In 263 SLOS patients 10 common alleles (c.964-1G>C, p.Trp151X, p.Thr93Met, p.Val326Leu, p.Arg352Trp, p.Arg404Cys, p.Phe302Leu, p.Leu157Pro, p.Gly410Ser, p.Arg445Gln) were found to constitute approximately 80% of disease-causing mutations. As reported before, the mutational spectra differed significantly between populations, and frequency peaks of common mutations were observed in North-West (c.964-1G>C), North-East (p.Trp151X, p.Val326Leu) and Southern Europe (p.Thr93Met). SLOS was virtually absent from Finland. The analysis of nearly 8000 alleles from 10 different European populations confirmed a geographical distribution of DHCR7 mutations as reported in previous studies. The common Null mutations in Northern Europe (combined ca. 1:70) occurred at a much higher frequency than expected from the reported prevalence of SLOS. In contrast the most common mutation in Mediterranean SLOS patients (p.Thr93Met) had a low population frequency. Haplotypes were constructed for SLOS chromosomes, and for wild-type chromosomes of African and European origins using eight cSNPs in the DHCR7 gene. The DHCR7 orthologue was sequenced in eight chimpanzees (Pan troglodytes) and three microsatellites were analysed in 50 of the SLOS families in order to estimate the age of the three major SLOS-causing mutations. CONCLUSIONS: The results indicate a time of first appearance of c.964-1G>C and p.Trp151X some 3000 years ago in North-West and North-East Europe, respectively. The p.Thr93Met mutations on the J haplotype has probably first arisen approximately 6000 years ago in the Eastern Mediterranean. Together, it appears that a combination of founder effects, recurrent mutations, and drift have shaped the present frequency distribution of DHCR7 mutations in Europe.

Mild Smith-Lemli-Opitz syndrome: further delineation of 5 Polish cases and review of the literature.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder caused by reduced activity of 7-dehydrocholesterol reductase, resulting in an increased concentrations of 7-dehydrocholesterol and 8-dehydrocholesterol in body fluids and tissues. Phenotypically it is characterized by wide range of abnormalities, from mild to lethal forms what causes difficulties in its clinical diagnostics. To further delineate the physical spectrum of the mild form of Smith-Lemli-Opitz syndrome, especially with regard to genotype-phenotype correlation, we describe 5 Polish patients with mild phenotype (one with novel mutation in DHCR7 gene and four published before) and analyze 18 other cases from the literature. As the conclusion we give recommendation for tests toward SLOS in cases with "idiopathic" intellectual impairment and/or behavioral anomalies, as well as in biochemically doubtful but clinically fitting cases with overall gestalt and history of this syndrome.

Detection of a deletion of exons 8-16 of the UBE3A gene in familial Angelman syndrome using a semi-quantitative dosage PCR based assay.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by failure of expression of the maternal copy of the imprinted UBE3A gene through a variety of mechanisms detected by methylation studies, mutation analysis of UBE3A and FISH. In 10-15% of suspected cases of AS these investigations do not reveal a genetic abnormality. We report here the development of a semi-quantitative dosage PCR technique used to identify sub-microscopic deletions involving UBE3A. Using this method we analysed a panel of 26 patients from 24 families, all fulfilling the clinical criteria for AS. We identified a deletion of UBE3A exons 8-16 in a sibling pair. Analysis of parental samples revealed the same deletion in their phenotypically normal mother. This is an inexpensive and valuable method for detecting UBE3A deletions in a small but important proportion of AS cases of unidentifiable cause.

SLOS carrier frequency in Poland as determined by screening for Trp151X and Val326Leu DHCR7 mutations.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis caused by mutations in the DHCR7 gene. Previous studies estimated the prevalence of SLOS between 1 in 10,000 to 1 in 70,358 based on case frequency surveys. Although panethnic, SLOS appears to be most frequent in Central European populations (Czech Republic 1 in 10,000, Slovakia 1 in 15,000 - 1 in 20,000). In Polish individuals with SLOS two DHCR7 mutations, c.452G>A (p.Trp151X) and c.976G>T (p.Val326Leu), account for 65.2% of all observed DHCR7 mutations. We analyzed 2169 samples for the p.Trp151X mutation and 2087 for the p.Val326Leu mutation. The combined carrier frequency of these two mutations of was 2.40+/-0.32%, yielding a calculated incidence of SLOS in Poland of 2.5 4x10(-4)-4.3 5x10(-4) (1 in 2,300 to 1 in 3,937) placing SLOS among the most common recessive genetic disorders in Poland.

Difficulties in recognition of pyruvate dehydrogenase complex deficiency on the basis of clinical and biochemical features. The role of next-generation sequencing.

Pyruvate dehydrogenase complex (PDHc) defect is a well-known cause of mitochondrial disorders (MD) with at least six responsible genes (PDHA1, PDHB, DLAT, DLD, PDHX, PDP1). The aim of this work was to assess the diagnostic value of biochemical methods in recognition of PDHc defect in Polish patients with suspicion of MD. In the first step, Western blot of the E1α subunit was performed on 86 archive muscle bioptates with suspicion of MD. In the second step, Sanger PDHA1 sequencing was performed in 21 cases with low E1α expression. In the third step, 7 patients with negative results of PDHA1 sequencing were subjected to whole-exome sequencing (WES). This protocol revealed 4 patients with PDHA1 and one with DLD mutations. Four additional probands were diagnosed outside the protocol (WES or Sanger sequencing). The molecular characterization of PDHc defect was conducted in a total of 9 probands: 5 according to and 4 off the protocol. Additionally, two affected relatives were recognized by a family study. Altogether we identified seven different PDHA1 changes, including two novel variants [c.464T > C (p.Met155Thr) and c.856_859dupACTT (p.Arg288Leufs*10)] and one DLD variant. The lactate response to glucose load in the PDHA1 subset was compared to a subset of non PDHc-related MD. Opposite responses were observed, with an increase of 23% and decrease of 27%, respectively. The results show that determining lactate response to glucose load and muscle E1α expression may contribute to distinguishing PDHc-related and other MD, however, WES is becoming the method of choice for MD diagnostics.

Identification of 14 novel mutations in DHCR7 causing the Smith-Lemli-Opitz syndrome and delineation of the DHCR7 mutational spectra in Spain and Italy.

The Smith-Lemli-Opitz syndrome (SLOS) is a phenotypically variable metabolic malformation and mental retardation syndrome for which more than 80 mutations in the DHCR7 disease-causing gene have been described. The DHCR7 mutational spectra differ significantly in different areas of Europe, and several common putative founder mutations account for a substantial fraction of all mutations in some ethnic groups. Here we have analysed 47 SLOS patients and describe 14 newly identified mutations in 18 SLOS patients of Ashkenazi Jewish, Austrian, British, German, Italian, Irish, Polish, Portuguese, and Spanish origins. Half of the new mutations are in the transmembrane domains of the protein. In addition, there were two null mutations, one mutation in the 4th cytoplasmic loop, two mutations in the first and last codons, and three mutations in other regions such as the second cytoplasmic loop and the first endoplasmic loop. The analysis included 20 Spanish and 12 Italian SLOS patients and revealed very different mutation spectra in these patients compared to previously described patients from Czechoslovakia, Germany, Poland, and the UK and implicated p.Thr93Met on the J haplotype as the most frequent Mediterranean founder mutation.

Maternal apo E genotype is a modifier of the Smith-Lemli-Opitz syndrome.

BACKGROUND: Smith-Lemli-Opitz syndrome (MIM 270400) is an autosomal recessive malformation and mental retardation syndrome that ranges in clinical severity from minimal dysmorphism and mild mental retardation to severe congenital anomalies and intrauterine death. Smith-Lemli-Opitz syndrome is caused by mutations in the Delta7 sterol-reductase gene (DHCR7; EC 1.3.1.21), which impair endogenous cholesterol biosynthesis and make the growing embryo dependent on exogenous (maternal) sources of cholesterol. We have investigated whether apolipoprotein E, a major component of the cholesterol transport system in human beings, is a modifier of the clinical severity of Smith-Lemli-Opitz syndrome. METHOD: Common apo E, DHCR7, and LDLR genotypes were determined in 137 biochemically characterised patients with Smith-Lemli-Opitz syndrome and 59 of their parents. RESULTS: There was a significant correlation between patients' clinical severity scores and maternal apo E genotypes (p = 0.028) but not between severity scores and patients' or paternal apo E genotypes. In line with their effects on serum cholesterol levels, the maternal apo epsilon2 genotypes were associated with a severe Smith-Lemli-Opitz syndrome phenotype, whereas apo E genotypes without the epsilon2 allele were associated with a milder phenotype. The correlation of maternal apo E genotype with disease severity persisted after stratification for DHCR7 genotype. There was no association of Smith-Lemli-Opitz syndrome severity with LDLR gene variation. CONCLUSIONS: These results suggest that the efficiency of cholesterol transport from the mother to the embryo is affected by the maternal apo E genotype and extend the role of apo E and its disease associations to modulation of embryonic development and malformations.

Parental mosaicism of JAG1 mutations in families with Alagille syndrome.

The Alagille syndrome (AGS), a congenital disorder affecting liver, heart, skeleton and eye in association with a typical face, is an autosomal dominant disease with nearly complete penetrance and variable expression. AGS is caused by mutations in the developmentally important JAG1 gene. In our mutation screening, where 61 mutations in JAG1 were detected, we identified five cases where mosaicism is present. Our results point to a significant frequency of mosaicism for JAG1 mutations in AGS of more than 8.2%. Because mosaicism may be associated with a very mild phenotype, the appropriate diagnosis of AGS and consequently the determination of the recurrence risk can be complicated.

Clinical ascertainment of Nijmegen breakage syndrome (NBS) and prevalence of the major mutation, 657del5, in three Slav populations.

Nijmegen breakage syndrome (NBS) is a chromosomal instability disorder, clinically characterised by microcephaly, immunodeficiency, radiosensitivity and a very high predisposition to lymphoid malignancy. Recently, it was demonstrated that mutations in the NBS1 gene are responsible for NBS. Most of the NBS patients known so far are of Slav origin and carry a major founder mutation 657del5 in exon 6 of the NBS1 gene. In this study we estimated the prevalence of the 657del5 mutation in the Czech Republic, Poland and the Ukraine. We found an unexpectedly high carrier frequency of the 657del5 mutation (1/177) in the three Slav populations, a factor that may contribute to cancer frequency in those countries. In addition, we show that NBS patients are often diagnosed late and therefore receive inappropriate therapy.

Frequency gradients of DHCR7 mutations in patients with Smith-Lemli-Opitz syndrome in Europe: evidence for different origins of common mutations.

Smith-Lemli-Opitz syndrome/RSH (SLOS) is a multiple congenital anomaly syndrome caused by mutations in the gene for Delta7-sterol reductase (DHCR7) which catalyses the last step in the biosynthesis of cholesterol. SLOS is among the common recessive disorders in Europeans but almost absent in most other populations. More than 40 mutations in the DHCR7 gene some of which are frequent have been described in SLOS patients of various origins. Here we report mutation analysis of the DHCR7 gene in SLOS patients from Poland (n = 15), Germany/Austria (n = 22) and Great Britain (n = 22). Altogether 35 different mutations were identified and the two null mutations IVS8-1G > C and W151X were the most frequent in the total sample. In all three populations three mutations accounted for >0.5 of SLOS chromosomes. The mutational spectra were, however, significantly different across these populations with each of the common mutations showing an east-west gradient (W151X, V326L) or vice versa (IVS8-1G > C). W151X is the most frequent (0.33) mutation in Polish SLOS patients. It has an intermediate frequency in German/Austrian patients (0.18) and is rare among British patients (0.02). V326L shows the same distribution pattern (Poland 0.23, Germany/Austria 0.18, Britain 0.02). In contrast IVS8-1G > C is most frequent in Britain (0.34) intermediate in Germany/Austria (0.20) and rare in Poland (0.03). All analysed IVS8-1G > C and V326L alleles shared the same DHCR7 haplotype, whereas the W151X mutation occurred on different haplotypes. There is evidence for both recurrent mutations and founder effects. Together this suggests that the common SLOS mutations in Europe have different geographic and historic origins and spread across the continent in opposite directions.