Genotype, phenotype and hormonal levels correlation in non-classical congenital adrenal hyperplasia.

Non-classical congenital adrenal hyperplasia (NCAH) is a morbid condition sustained by the reduced function of one of the enzymes involved in the adrenal steroid biosynthesis pathway, mainly the 21-hydroxylase. Different degrees of enzyme activity impairment determine different clinical pictures, with childhood or post-pubertal onset. The aim of this study was to evaluate the relationship between genotype, phenotype, and adrenal hormonal levels in a group of 66 patients affected by NCAH attending outpatient pediatric or endocrinological Clinics. Our findings show that age at pubarche/menarche was significantly younger, height SD score) and Δ bone age-chronological age were significantly higher in patients with a more severe enzyme activity impairment, while cutaneous androgenization and menstrual irregularities in post-pubertal girls were not related to the grading of genotype.

A family with autosomal dominant leukodystrophy linked to 5q23.2-q23.3 without lamin B1 mutations.

BACKGROUND AND PURPOSE: Duplications of lamin B1 (LMNB1) at 5q23 are implicated in adult-onset autosomal dominant leukodystrophy (ADLD) having been described in six families with diverse ethnic background but with a homogeneous phenotype. In a large Italian family, we recently identified a variant form of ADLD characterized clinically by absence of the autonomic dysfunction at onset described in ADLD and, on MRI, by milder cerebellar involvement with sparing of hemispheric white matter. Aim of this study was to investigate the genetic basis of this variant form of ADLD. METHODS: We carried out a genome-wide linkage analysis using microsatellite markers, and the genes in the candidate region were screened for point mutations. LMNB1 was also screened for deletions/duplications by real-time PCR, multiplex ligation-dependent probe amplification and Southern blot. RESULTS: We mapped the variant ADLD locus to 5q23.2-q23.3, a genomic region containing 11 genes including LMNB1. Neither gene copy-number defects nor point mutations in the LMNB1 gene were found. We also excluded point mutations in the coding exons of the other ten genes in the candidate region. However, expression of lamin B1 evaluated in lymphoblastoid cells was higher in patients than in healthy controls, and was similar to the lamin B1 expression levels found in a patient with LMNB1 duplication. CONCLUSIONS: This observation suggests that a mutation in an LMNB1 regulatory sequence underlies the variant ADLD phenotype. Thus, adult forms of ADLD linked to 5q23 appear to be more heterogeneous clinically and genetically than previously thought.

Mutations in the lamin B1 gene are not present in multiple sclerosis.

BACKGROUND: Whole gene duplication of the lamin B1 gene (LMNB1), encoding for a protein of the nuclear lamina, causes an adult-onset autosomal dominant leukodystrophy (ADLD). Clinical features of ADLD (onset in adult life, dysautonomic symptoms, followed by pyramidal and cerebellar dysfunctions) partially resemble those of multiple sclerosis (MS), particularly the primary-progressive form. Our aim was to test whether LMNB1 gene mutations were present amongst patients with a diagnosis of MS. METHODS: One hundred eighty-two MS patients were screened for copy number variations of the LMNB1 gene using a qPCR assay. Point mutations in the LMNB1 gene were searched by denaturing high-performance liquid chromatography and direct sequencing in a subgroup of 16 patients with familial MS. RESULTS: No duplication/deletion of the lamin B1 gene was found amongst MS patients, and no point mutation was identified in the familial cases. CONCLUSION: Our work indicates that lamin B1 defects are probably not responsible for signs and symptoms resembling multiple sclerosis.

A novel family with Lamin B1 duplication associated with adult-onset leucoencephalopathy.

BACKGROUND AND AIMS: Duplication of the lamin B1 gene (LMNB1) has recently been described in a rare form of autosomal dominant adult-onset leucoencephalopathy. The aim of the study was to evaluate the presence of LMNB1 gene defects in a series of eight patients with diffuse adult-onset hereditary leucoencephalopathy. METHODS: Clinical features of tested patients included a variable combination of pyramidal, cerebellar, cognitive and autonomic dysfunction. Neuroradiological data (MRI) showed symmetrical and diffuse white-matter lesions in six cases, and multifocal confluent lesions in two. LMNB1 full gene deletion/duplication and point mutations were searched using a TaqMan real-time PCR assay and direct sequencing of all coding exons. RESULTS: One patient carried a 140-190 kb duplication involving the entire LMNB1 gene, the AX748201 transcript and the 3' end of the MARCH3 gene. Clinical and neuroimaging data of this proband and an affected relative overlapped with the features already described in patients with LMNB1 duplication. Lamin B1 expression was found increased in lymphoblasts. No LMNB1 gene defect was identified in the remaining seven probands. CONCLUSIONS: LMNB1 gene duplication appears characteristic of a subset of adult-onset autosomal dominant leucoencephalopathies, sharing autonomic dysfunction at onset, diffuse T2-hyperintensity of supra- and infratentorial white matter, sparing of U-fibres and optic radiations. The variable phenotypes in the remaining cases lacking LMNB1 defects (five with autosomal dominant transmission) suggest that adult-onset leucoencephalopathies are genetically heterogeneous.

The polymorphic polyglutamine repeat in the mitochondrial DNA polymerase gamma gene is not associated with oligozoospermia.

The POLG1 nuclear gene, encoding for the catalytic subunit of the mitochondrial polymerase gamma, has been reported to play a role in male infertility. In fact, genotypes showing alleles different from the common ten repeat CAG allele have been detected in patients with oligozoospermia or in patients with normal spermiograms and unexplained infertility. However, these results have been debated by other studies. To verify these data, we analyzed 625 individuals in three groups of case-controls from three different Italian regions. In these series, the frequency of the different genotypes was not statistically different in oligozoospermic vs normal subjects. Even considering the pooled controls and patients (348 and 277, respectively), no significant difference was shown (p = 0.11). Our findings, in agreement with other studies from Italy and France, suggest that, at least in these countries, the POLG1 CAG-repeat polymorphisms do not contribute to oligozoospermia.

Camurati-Engelmann disease: review of the clinical, radiological, and molecular data of 24 families and implications for diagnosis and treatment.

Camurati-Engelmann disease (CED) is a rare autosomal dominant type of bone dysplasia. This review is based on the unpublished and detailed clinical, radiological, and molecular findings in 14 CED families, comprising 41 patients, combined with data from 10 other previously reported CED families. For all 100 cases, molecular evidence for CED was available, as a mutation was detected in TGFB1, the gene encoding transforming growth factor (TGF) beta1. Pain in the extremities was the most common clinical symptom, present in 68% of the patients. A waddling gait (48%), easy fatigability (44%), and muscle weakness (39%) were other important features. Radiological symptoms were not fully penetrant, with 94% of the patients showing the typical long bone involvement. A large percentage of the patients also showed involvement of the skull (54%) and pelvis (63%). The review provides an overview of possible treatments, diagnostic guidelines, and considerations for prenatal testing. The detailed description of such a large set of CED patients will be of value in establishing the correct diagnosis, genetic counselling, and treatment.

FMR1 gene premutation is a frequent genetic cause of late-onset sporadic cerebellar ataxia.

In an Italian population of 275 unrelated men affected by adult-onset sporadic progressive cerebellar ataxia, the authors found six patients carrying an FMR1 gene premutation. Age at onset (range, 53 to 69 years) and clinical-neuropathologic findings were consistent with the fragile-X tremor ataxia syndrome (FXTAS), although tremor was not as common as previously described. FXTAS accounted for 4.2% of the cases diagnosed at >50 years, suggesting that it is a frequent genetic cause of late-onset sporadic ataxia.

TSC1 and TSC2 deletions differ in size, preference for recombinatorial sequences, and location within the gene.

Large TSC gene rearrangements are not rare findings in tuberous sclerosis. Interestingly, all deletions, duplications and inversions so far described involve TSC2, none being associated with TSC1. In order to shed light on the structural basis of the preferential DNA rearrangements in TSC2 over TSC1 and to assess, in an unselected patient population, the prevalence of large re-arrangements in both TSC loci, we screened 202 tuberous sclerosis patients consecutively referred at our center. Southern blot analysis on EcoRI+HindIII double-digested DNA identified 19 partial or full-length gene deletions: three involved TSC1 and sixteen TSC2. The breakpoint sequence of seven internal deletions, three in TSC1 and four in TSC2, allowed us to speculate on the mechanism favoring TSC2 unequal recombinations and to identify a deletion hot spot that lies in TSC1 and that may be relevant in the routine genetic testing of tuberous sclerosis. Briefly, three major features appear to distinguish TSC1 from TSC2 deletions: (1) deletion size: all TSC1 deletions are within the transcriptional unit, whereas 12 of the 16 TSC2 deletions have at least one external breakpoint; (2) location within the gene: all TSC1 deletions are confined to the 3'end of the gene (all three 5' breakpoints being located in intron 20) thus resulting in the same frameshift mutation following amino acid K875, whereas the TSC2 internal breakpoints appear to be scattered along the gene; (3) preference for recombinatorial sequences: six out of eight internal TSC2 breakpoints map within Alu repeats, whereas none of the three TSC1 deletions appear to be Alu-mediated. Indeed, in the latter gene, unique structural features (a purine-rich tract flanked by pyrimidine-rich segments) surrounding one of the two identified breakpoint cluster regions might play a role in promoting inappropriate recombinations.

Mutations in the gene encoding the latency-associated peptide of TGF-beta 1 cause Camurati-Engelmann disease.

Camurati-Engelmann disease (CED; MIM 131300), or progressive diaphyseal dysplasia, is a rare, sclerosing bone dysplasia inherited in an autosomal dominant manner. Recently, the gene causing CED has been assigned to the chromosomal region 19q13 (refs 1-3). Because this region contains the gene encoding transforming growth factor-beta 1 (TGFB1), an important mediator of bone remodelling, we evaluated TGFB1 as a candidate gene for causing CED.

Localisation of the gene causing diaphyseal dysplasia Camurati-Engelmann to chromosome 19q13.

Camurati-Engelmann disease, progressive diaphyseal dysplasia, or diaphyseal dysplasia Camurati-Engelmann is a rare, autosomal dominantly inherited bone disease, characterised by progressive cortical expansion and sclerosis mainly affecting the diaphyses of the long bones associated with cranial hyperostosis. The main clinical features are severe pain in the legs, muscular weakness, and a waddling gait. The underlying cause of this condition remains unknown. In order to localise the disease causing gene, we performed a linkage study in a large Jewish-Iraqi family with 18 affected subjects in four generations. A genome wide search with highly polymorphic markers showed linkage with several markers at chromosome 19q13. A maximum lod score of 4.9 (theta=0) was obtained with markers D19S425 (58.7 cM, 19q13.1) and D19S900 (67.1 cM, 19q13. 2). The disease causing gene is located in a candidate region of approximately 32 cM, flanked by markers D19S868 (55.9 cM, 19q13.1) and D19S571 (87.7 cM, 19q13.4).

The SH3 domains of endophilin and amphiphysin bind to the proline-rich region of synaptojanin 1 at distinct sites that display an unconventional binding specificity.

The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the "classical" type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.

Polycystin-1 expression in PKD1, early-onset PKD1, and TSC2/PKD1 cystic tissue.

BACKGROUND: The mutational mechanism responsible for cyst formation in polycystic kidney disease 1 gene (PKD1) remains controversial, with data indicating a two-hit mechanism, but also evidence of polycystin-1 expression in cystic tissue. METHODS: To investigate this apparent paradox, we analyzed polycystin-1 expression in cystic renal or liver tissue from 10 patients with truncating PKD1 mutations (including one early-onset case) and 2 patients with severe disease associated with contiguous deletions of TSC2 and PKD1, using monoclonal antibodies (mAbs) to both extreme N-(7e12) and C-terminal (PKS-A) regions of the protein. Truncation of the C-terminal epitope from the putative mutant proteins in each case allowed exclusive assessment of the nontruncated protein with PKS-A. RESULTS: In adult PKD1 tissue, the majority of cysts (approximately 80%) showed polycystin-1 expression, although staining was absent in a variable but significant minority (approximately 20%), in spite of the normal expression of marker proteins. Unlike adult PKD1, however, negative cysts were rarely found in infantile PKD1 or TSC2/PKD1 deletion cases. CONCLUSIONS: If a two-hit mutational mechanism is operational, these results suggest that the majority of somatic mutations in adult PKD1 are likely to be missense changes. The low level of polycystin-1-negative cysts in the three "early-onset" cases, however, suggests that a somatic PKD1 mutation may not always be required for cyst formation.

A large TSC2 and PKD1 gene deletion is associated with renal and extrarenal signs of autosomal dominant polycystic kidney disease.

BACKGROUND: The renal lesions in tuberous sclerosis complex (TSC) consist in multiple angiomyolipomas, often associated with cysts of variable size. Recently a few TSC patients with early-onset renal cysts resembling the autosomal dominant polycystic kidney disease (ADPKD) have been described. Virtually all of them showed deletions of both TSC2 and PKD1 genes. METHODS: Two unrelated families in which TSC and PKD co-segregate were investigate. 16p13.3-linked haplotype segregation, Southern blot, pulsed field gel electrophoresis, and loss of heterozygosity analyses were performed in both affected and unaffected family members. RESULTS: The proband from family 1 was first recognized as presenting typical neurological signs and skin lesions of TSC and multiple renal cysts at 12 years of age. Haemodialysis became necessary at age 28. CT and MRI scans revealed multiple cysts in the live and an asymptomatic, 3-4 mm aneurysm of the middle cerebral artery. His mother, who died at 47 of breast cancer, had ADPKD and reached the ESRD at 42. She showed facial angiofibromas. Both patients carried a submicroscopic germline deletion spanning the entire TSC2 gene and the large majority of PKD1 coding sequence. In the proband from family 2, the TSC diagnosis was made at 4 years. Enlarged polycystic kidneys causing and-stage renal failure at 19 years were observed. This patient carried a large germline, de novo deletion involving the entire TSC2 and PKD1 genes. In addition we could show in a renal hamartoma from this subject the loss of heterozygosity of markers spanning the TSC2 and PKD1 genes from the residual, normal chromosome 16 of paternal origin. CONCLUSIONS: The presence of a deletion involving both TSC2 and PKD1 genes should be considered in the clinical assessment of TSC children with an early-onset polycystic kidney disease, and more generally in all ADPKD patients who develop end-stage renal failure prior to the fourth or fifth decade of life. Finally, the occurrence of typical renal and extrarenal signs of ADPKD in a PKD1 hemizygote individual seems to support concept that a somatic inactivation of the residual PKD1 gene is required for the development of the cysts.

A novel SH3-containing human gene family preferentially expressed in the central nervous system.

The Src-homology-3 domain (SH3) is an evolutionarily conserved, 50- to 60-amino-acid module carried by intracellular proteins involved in the transduction of signals for cell polarization, motility, enzymatic activation, and transcriptional regulation. The SH3 drives protein-protein interactions through binding to proline-rich ligands. This function relies on the conserved secondary structure, whereas the SH3 primary structure is highly diverse. Taking advantage of the fact that the few conserved amino acids are clustered near the N- and C-terminal ends, we designed degenerate oligonucleotides spanning these two regions and screened by PCR a variety of normal and tumor tissues for the expression of SH3-containing transcripts. Using this strategy, we have identified a novel SH3-containing human gene family of six related transcripts that map to four different chromosomes. The SH3 domain lies at the C-terminal end and shows 56-50% amino acid homology to the C-terminal SH3 of Sem-5/Drk/GRB2. The N-terminal segment of this novel SH3GL (from SH3-containing Grb2-like) gene family does not resemble any known protein. Three of these transcripts are in-frame and show a peculiar tissue distribution: SH3GL2 is preferentially expressed in the brain, SH3GL3 in brain and testis, and SH3GL1 is ubiquitous.

Tuberous sclerosis complex: neonatal deaths in three of four children of consanguineous, non-expressing parents.

We describe here four sibs, born to consanguineous, healthy, asymptomatic parents. Three of these infants had a rapidly fatal course in the neonatal period; death was attributed to congestive heart failure with radiographic evidence of cardiomegaly in all of them. Necropsy was done in only one of them and showed the typical findings of tuberous sclerosis complex (TSC) in the central nervous system (CNS), kidneys, heart, and liver. The fourth sib, currently 2 years old, also has typical signs of TSC, namely hypomelanotic skin macules and calcified subependymal nodules. Both parents and a living maternal grandmother had appropriate examination, which included skin inspection under Wood's lamp, dental examination, fundoscopy, echocardiography, abdominal and renal ultrasound, and head CT and MRI scans, and no signs of TSC were found in either parent or in the only living grandmother. By history alone there is no other relative with signs or symptoms suggestive of TSC. Linkage analysis and loss of heterozygosity (LOH) investigations on a variety of lesions obtained from postmortem and tissue or blood specimens from all available family members studied failed to identify a microdeletion in the chromosomal regions where TSC genes are located. It is very unusual that in a single TSC family there were three consecutive neonatal deaths, and very likely that all had cardiac rhabdomyomas. Moreover, to the best of our knowledge, there are no previous reports of TSC families with more than one affected sib, unusually severe manifestations of the disease, and completely normal, consanguineous parents.