Arie P.T. Smits

Heterozygous Germline Mutations in the p53 Homolog p63 Are the Cause of EEC Syndrome

EEC syndrome is an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts. We have mapped the genetic defect in several EEC syndrome families to a region of chromosome 3q27 previously implicated in the EEC-like disorder, limb mammary syndrome (LMS). Analysis of the p63 gene, a homolog of p53 located in the critical LMS/EEC interval, revealed heterozygous mutations in nine unrelated EEC families. Eight mutations result in amino acid substitutions that are predicted to abolish the DNA binding capacity of p63. The ninth is a frameshift mutation that affects the p63alpha, but not p63beta and p63gamma isotypes. Transactivation studies with these mutant p63 isotypes provide a molecular explanation for the dominant character of p63 mutations in EEC syndrome.

Rapid antibody test for diagnosing fragile X syndrome : a validation of the technique

Abstract To date, the identification of patients and carriers of the fragile X syndrome has been carried out by DNA analysis by means of the polymerase chain reaction and Southern blot analysis. This direct DNA analysis allows both the size of the CGG repeat and methylation status of the FMR1 gene to be determined. We have recently presented a rapid antibody test on blood smears based on the presence of FMRP, the protein product of the FMR1 gene, in lymphocytes from normal individuals and the absence of FMRP in lymphocytes from patients. Here, we have tested the diagnostic value of this new technique by studying FMRP expression in 173 blood smears from normal individuals and fragile X patients. The diagnostic power of the antibody test is “perfect” for males, whereas the results are less specific for females.

Imprinting Effect in Premature Ovarian Failure Confined to Paternally Inherited Fragile X Premutations

Fragile X premutations are considered to be a risk factor for premature ovarian failure (POF), which is usually defined as menopause at age <40 years. Since premutations may be inherited from either the mother or the father, we evaluated the influence of the inheritance pattern on the duration of reproductive life in female carriers. The occurrence of POF and age at menopause in women with a paternally inherited fragile X premutation (PIP) were compared to those in women with a maternally inherited fragile X premutation (MIP). We identified 148 women in whom the parental origin of the premutation could be determined. In 109 of these women we were able to establish whether POF had occurred: 82 women had a PIP, and 27 had a MIP. Twenty-three of the women (28%) with a PIP had POF, versus only 1 (3.7%) with a MIP (two -tailed Fishers exact test; P=. 007). Kaplan-Meier analysis of all 148 premutations showed that the age at menopause was significantly lower in the women with a PIP than in the woman with a MIP (Breslow test in Kaplan-Meier analysis; P=.003). Our data strongly suggest that, when POF occurs in fragile X premutation carriers, a considerable proportion of the premutations are inherited paternally (parent-of-origin effect). We hypothesize that this may be owing to a paternal genomic imprinting effect.

Widespread non-central nervous system organ pathology in fragile X premutation carriers with fragile X-associated tremor/ataxia syndrome and CGG knock-in mice

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder generally presenting with intention tremor and gait ataxia, but with a growing list of co-morbid medical conditions including hypothyroidism, hypertension, peripheral neuropathy, and cognitive decline. The pathological hallmark of FXTAS is the presence of intranuclear inclusions in both neurons and astroglia. However, it is unknown to what extent such inclusions are present outside the central nervous system (CNS). To address this issue, we surveyed non-CNS organs in ten human cases with FXTAS and in a CGG repeat knock-in (CGG KI) mouse model known to possess neuronal and astroglial inclusions. We find inclusions in multiple tissues from FXTAS cases and CGG KI mice, including pancreas, thyroid, adrenal gland, gastrointestinal, pituitary gland, pineal gland, heart, and mitral valve, as well as throughout the associated autonomic ganglia. Inclusions were observed in the testes, epididymis, and kidney of FXTAS cases, but were not observed in mice. These observations demonstrate extensive involvement of the peripheral nervous system and systemic organs. The finding of intranuclear inclusions in non-CNS somatic organ systems, throughout the PNS, and in the enteric nervous system of both FXTAS cases as well as CGG KI mice suggests that these tissues may serve as potential sites to evaluate early intervention strategies or be used as diagnostic factors.

Female carriers of fragile X premutations have no increased risk for additional diseases other than premature ovarian failure

Carriers of fragile X premutations were previously considered phenotypically normal but are now known to be at risk for premature ovarian failure (POF). This prompted us to investigate whether premutation carriers (PC) have an increased risk for other diseases. We approached 306 women out of 84 fra(X) families of whom 264 (86.3%) participated in this study. A medical history was taken of these women. Whenever possible, the anamnestic data were verified with medical records. We first evaluated the occurrence of diseases that are commonly associated with menopause in PC and compared this to that in women with either a normal FMR‐1 gene or a full mutation. We found no statistically significant differences in the occurrence of diseases known to be associated with menopause, such as cardiovascular diseases and osteoporosis. However, lower bone mineral density was observed only in PC. Subsequently, we compared the occurrence of other medical problems between the two groups by estimating relative risks. PC did not demonstrate other diseases more commonly compared to non‐PC from the same families. These findings are important for the counseling of PC. Carriership of the premutation may affect the ovaries, but does not substantially increase the risk for additional medical problems. Once a PC does experience POF, she is at risk for early estrogen deprivation, which may lead to a premature decrease in bone density, when not treated.

Molecular analysis of a familial case of renal cell cancer and a t(3;6)(q12;q15).

We identified a novel familial case of clear‐cell renal cancer and a t(3;6)(q12;q15). Subsequent cytogenetic and molecular analyses showed the presence of several abnormalities within tumour samples obtained from different patients. Loss of the der(3) chromosome was noted in some, but not all, of the samples. A concomitant VHL gene mutation was found in one of the samples. In addition, cytogenetic and molecular evidence for heterogeneity was obtained through analysis of several biopsy samples from one of the tumours. Based on these results and those reported in the literature, we conclude that loss of der(3) and subsequent VHL gene mutation may represent critical steps in the development of renal cell cancers in persons carrying the chromosome 3 translocation. Moreover, preliminary data suggest that other (epi)genetic changes may be related to tumour initiation.

A new locus for postaxial polydactyly type A/B on chromosome 7q21-q34.

Postaxial polydactyly (PAP) is the occurrence of one or more extra ulnar or fibular digits or parts of it. In PAP-A, the extra digit is fully developed and articulates with the fifth or an additional metacarpal/metatarsal, while it is rudimentary in PAP-B. Isolated PAP usually segregates as an autosomal dominant trait, with variable expression. Three loci are known for PAP in humans. PAPA1 (including PAP-A/B in one patient) on 7p13 caused by mutations in the GLI3 gene, PAPA2 on 13q21–q32 in a Turkish kindred with PAP-A only, and a third one (PAPA3) in a Chinese family with PAP-A/B on 19p13.1–13.2. We identified a fourth locus in a large Dutch six-generation family with 31 individuals including 11 affecteds. Their phenotype varied from either PAP-A, or PAP-B to PAP-A/B with or without the co-occurence of partial cutaneous syndactyly. We performed a whole-genome search and found linkage between PAP and markers on chromosome 7q. The highest LOD score was 3.34 obtained at D7S1799 and D7S500 with multipoint analysis.

Intra-individual stability over time of standardized anti-Müllerian hormone in FMR1 premutation carriers

BACKGROUND Carriers of a premutation (CGG repeat length 55-200) in the fragile X mental retardation (FMR1) gene are at risk for primary ovarian insufficiency (FXPOI). The anti-Müllerian hormone (AMH) level acts as a useful marker of ovarian follicle reserve and, thus, may serve to predict when this ovarian reserve becomes too low to sustain ovarian function. We investigated the intra-individual variation of AMH levels over time for premutation carriers compared with non-carriers. METHODS We determined AMH levels in blood samples from 240 women ascertained through fragile X families, of which 127 were premutation carriers and 113 were non-carriers. Linear mixed models were used to assess the effect of age and premutation status on AMH levels and to determine a modeled AMH value. The stability over time of the deviation of observed AMH levels from modeled levels, referred to as standardized AMH values, was assessed through correlation coefficients of 41 longitudinal samples. RESULTS At all ages, premutation carriers exhibited lower AMH levels. For all women, AMH was found to decrease by 10% per year. The added effect of having a premutation decreased AMH levels by 54%. The deviation of an individuals AMH level from the modeled value showed a reasonable intra-individual correlation. The Pearson correlation coefficient of two samples taken at different ages was 0.36 (P = 0.05) for non-carriers and 0.69 (P = 0.01) for carriers. CONCLUSIONS We developed a unique standardized AMH value, taking FMR1 premutation status and the subjects age into account, which appears to be stable over time and may serve as a predictor for FXPOI after further longitudinal assessment.

Menstrual disorders and endocrine profiles in fragile X carriers prior to 40 years of age: a pilot study.

This prospective study reports serum hormone measurements associated with menopause in fragile X carriers (n = 9, age between 31-40 years). These results demonstrate the occurrence of premature ovarian failure.

Four families (MRX43, MRX44, MRX45, MRX52) with nonspecific X-linked mental retardation: clinical and psychometric data and results of linkage analysis.

Four families are described in which mental retardation segregates in an X-linked fashion. Mental retardation was the only consistent clinical finding in all affected males. The degree of retardation varied from mild to profound both between and within families. Linkage analysis localized the genetic defect of MRX43 to Xp22. 31-p21.2, MRX44 to Xp11.3-p11.21, MRX45 to Xp11.3-p11.21, and MRX52 to Xp11.21-q21.33 with LOD scores of >2 at straight theta = 0.0 in all four families.