Schimke immuno-osseous dysplasia: a clinicopathological correlation

Background

Schimke immuno‐osseous dysplasia (SIOD) is a fatal autosomal recessive disorder caused by loss‐of‐function mutations in swi/snf‐related matrix‐associated actin‐dependent regulator of chromatin, subfamily a‐like 1 (SMARCAL1).

Methods

Analysis of detailed autopsies to correlate clinical and pathological findings in two men severely affected with SIOD.

Results

As predicted by the clinical course, T cell deficiency in peripheral lymphoid organs, defective chondrogenesis, focal segmental glomerulosclerosis, cerebral ischaemic lesions and premature atherosclerosis were identified. Clinically unexpected findings included a paucity of B cells in the peripheral lymphoid organs, emperipolesis‐like (penetration of one cell by another) abnormalities in the adenohypophysis, fatty infiltration of the cardiac right ventricular wall, pulmonary emphysema, testicular hypoplasia with atrophy and azospermia, and clustering of small cerebral vessels.

Conclusions

A regulatory role for the SMARCAL1 protein in the proliferation of chondrocytes, lymphocytes and spermatozoa, as well as in the development or maintenance of cardiomyocytes and in vascular homoeostasis, is suggested. Additional clinical management guidelines are recommended as this study has shown that patients with SIOD may be at risk of pulmonary hypertension, combined immunodeficiency, subcortical ischaemic dementia and cardiac dysfunction.The osteochondrodysplasias are a heterogeneous group of inherited disorders of skeletal growth causing disproportionate short stature. Of the 230 distinct osteochondrodysplasias,² several have been associated with nephrotic syndrome or immunodeficiency.² Schimke immuno‐osseous dysplasia (SIOD), an autosomal recessive panethnic multisystem osteochondrodysplasia, is characterised by dysmorphism,^3,4 spondyloepiphysial dysplasia,^5,6 T cell immunodeficiency^3,5 and nephrotic syndrome.^{3,5,7,8,9,10,11} Less penetrant features include hypothyroidism,³ migraine‐like headaches,¹² cerebral ischaemia^7,13 and enteropathy.^14,15,16,17 Stroke, severe opportunist infections, bone marrow failure, complications of renal failure or an undefined pulmonary disease,³ result in premature mortality in most patients during childhood to early adolescence; however, a few patients remain alive in their third and fourth decades of life.¹⁸SIOD is caused by biallelic loss‐of‐function mutations in swi/snf‐related matrix‐associated actin‐dependent regulator of chromatin, subfamily a‐like 1 (SMARCAL1).¹⁹ Previous studies have shown that SMARCAL1 encodes a protein homologous to the SNF2 family of chromatin remodelling proteins.^20,21 However, the mechanism by which SMARCAL1 mutations cause SIOD remains unknown. Hypotheses on the pathophysiology have considered SIOD to be an autoimmune disorder,^5,14 a connective tissue disorder,^16,22,23 a vascular endothelial disorder,^7,8,24 a metabolic disorder affecting chondrocyte and T cell differentiation,^5,16 or a cellular proliferation disorder.^3,19Murine Smarcal1 is expressed in tissues equivalent to those implicated as affected by clinical symptoms.²⁵ From this observation and clinical data, we have hypothesised that SIOD arises autonomously in each cell type. To determine whether disease occurs in each of these tissues as hypothesised and to elucidate further the pathophysiology of SIOD and potential functions for SMARCAL1, we undertook a detailed analysis of autopsy samples from two patients with SIOD and correlated the findings with their clinical course. Although secondary effects may confound interpretation of some findings, we find that the loss of functional SMARCAL1 has tissue‐specific effects on cellular proliferation, development and maintenance, and uncovered previously unknown pathological features relevant for management of patients with SIOD.     

Three single-nucleotide polymorphisms in LPA account for most of the increase in lipoprotein(a) level elevation in African Americans compared with European Americans

Background

The extent which universally common or population‐specific alleles can explain between‐population variations in phenotypes is unknown. The heritable coronary heart disease risk factor lipoprotein(a) (Lp(a)) level provides a useful case study of between‐population variation, as the aetiology of twofold higher Lp(a) levels in African populations compared with non‐African populations is unknown.

Objective

To evaluate the association between LPA sequence variations and Lp(a) in European Americans and African Americans and to determine the extent to which LPA sequence variations can account for between‐population variations in Lp(a).

Methods

Serum Lp(a) and isoform measurements were examined in 534 European Americans and 249 African Americans from the Choices for Healthy Outcomes in Caring for End‐Stage Renal Disease Study. In addition, 12 LPA variants were genotyped, including 8 previously reported LPA variants with a frequency of >2% in European Americans or African Americans, and four new variants.

Results

Isoform‐adjusted Lp(a) level was 2.23‐fold higher among African Americans. Three single‐nucleotide polymorphisms (SNPs) were independently associated with Lp(a) level (p<0.02 in both populations). The Lp(a)‐increasing SNP (G‐21A, which increases promoter activity) was more common in African Americans, whereas the Lp(a)‐lowering SNPs (T3888P and G+1/inKIV‐8A, which inhibit Lp(a) assembly) were more common in European Americans, but all had a frequency of <20% in one or both populations. Together, they reduced the isoform‐adjusted African American Lp(a) increase from 2.23 to 1.37‐fold(a 60% reduction) and the between‐population Lp(a) variance from 5.5% to 0.5%.

Conclusions

Multiple low‐prevalence alleles in LPA can account for the large between‐population difference in serum Lp(a) levels between European Americans and African Americans.Most genetic association studies focus on within‐population, rather than between‐population, differences in disease susceptibility. Although between‐population variation accounts for only 5–15% of human genetic diversity,¹ it is invoked increasingly to explain differences in disease risk and drug response across populations.^2,3 It is unknown whether such differences result from universally common alleles, which are hypothesised to contribute to within‐population risk variation for common diseases,^4,5 or from alleles that are population specific or nearly so. The allelic spectrum of between‐population variation has important implications, as identifying susceptibility alleles that are population specific may require strategies different from those identifying alleles that are common in all populations.The coronary heart disease risk factor lipoprotein(a) (Lp(a)) level is twofold higher in African than in non‐African populations,^6,7 and provides a useful case study of between‐population variation. The cardiovascular pathogenicity of Lp(a) is established best in Caucasians^8,9 and probably involves the low‐density lipoprotein‐bound plasminogen homologue apolipoprotein(a) (apo(a)), may increase low‐density lipoprotein delivery¹⁰ and may inhibit plasminogen‐mediated thrombolysis.¹¹ The apo(a) gene (LPA; MIM 152200) explains about 90% and 80% of Lp(a) level variance in European American¹² and African American¹³ populations, respectively. A genomically unusual 5.6‐kb variable tandem repeat in LPA encodes the KIV‐2 units, whose number determines apo(a) isoform size and explains half of the LPA effect on Lp(a) level.^12,14 The inverse association between isoform size and Lp(a) level probably reflects impaired cellular secretion of larger isoforms, which has been observed in vitro.¹⁵Isoform distributions are similar across populations, and do not explain higher African Lp(a) levels.⁷ Eight LPA variants (all but one are single‐nucleotide polymorphisms (SNPs)) have been associated with the isoform‐adjusted Lp(a) level in Europeans^{16,17,18,14,19,20} or Africans.^21,18 Some investigators have speculated that unidentified trans‐acting^16,22 or environmental²³ factors may explain the between‐population difference, as none of these have been shown to contribute substantially to higher African Lp(a) levels. . However, a substantial contribution of LPA to the between‐population difference cannot be excluded and provides the simplest explanation.Previous analyses have not considered multiple LPA variants simultaneously or comprehensively, and the extensive linkage disequilibrium across the gene^15,18,14,19 is expected to confound single‐locus effect estimates. Also, few studies included both European and African or African American populations, precluding direct quantification of LPA variant contributions to the between‐population difference. We report the results of a simultaneous analysis of multiple LPA variants, isoforms and Lp(a) level in a cohort of European Americans and African Americans.     

Array-based comparative genomic hybridisation identifies high frequency of cryptic chromosomal rearrangements in patients with syndromic autism spectrum disorders

Background

Autism spectrum disorders (ASD) refer to a broader group of neurobiological conditions, pervasive developmental disorders. They are characterised by a symptomatic triad associated with qualitative changes in social interactions, defect in communication abilities, and repetitive and stereotyped interests and activities. ASD is prevalent in 1 to 3 per 1000 people. Despite several arguments for a strong genetic contribution, the molecular basis of a most cases remains unexplained. About 5% of patients with autism have a chromosome abnormality visible with cytogenetic methods. The most frequent are 15q11–q13 duplication, 2q37 and 22q13.3 deletions. Many other chromosomal imbalances have been described. However, most of them remain undetectable using routine karyotype analysis, thus impeding diagnosis and genetic counselling.

Methods and results

29 patients presenting with syndromic ASD were investigated using a DNA microarray constructed from large insert clones spaced at approximately 1 Mb intervals across the genome. Eight clinically relevant rearrangements were identified in 8 (27.5%) patients: six deletions and two duplications. Altered segments ranged in size from 1.4 to 16 Mb (2–19 clones). No recurrent abnormality was identified.

Conclusion

These results clearly show that array comparative genomic hybridisation should be considered to be an essential aspect of the genetic analysis of patients with syndromic ASD. Moreover, besides their importance for diagnosis and genetic counselling, they may allow the delineation of new contiguous gene syndromes associated with ASD. Finally, the detailed molecular analysis of the rearranged regions may pave the way for the identification of new ASD genes.Autism spectrum disorders (ASD) belong to the group of pervasive developmental disorders (PDD). According to the Diagnostic statistical manual for mental disorders—fourth edition (DSM IV) classification,¹ ASD are characterised by impairments in communication, social skills and restricted or stereotyped pattern of behaviours and interests. A diagnosis within the autism spectrum requires one or more symptoms in each of the three areas of impairment. The prevalence of ASD is estimated at about 1/1000 to 3/1000.^2,3 ASD are heterogeneous conditions which can be either isolated or syndromic—that is, associated with other clinical features such as facial dysmorphism, limb or visceral malformations, and growth abnormalities.A total of 10–20% of ASD cases are due to known medical conditions involving chromosomal imbalances, genetic disorders (X fragile syndrome and tuberous sclerosis)⁴ or environmental factors (valproate⁵ and rubella). The other cases remain unexplained. Twin and familial studies have documented a higher concordance rate in monozygotic twins (90%) than in dizygotic twins (4.5%),^6,7,8 and a 75‐fold greater risk to siblings in idiopathic patients than in the general population.^9,10 Collectively, these studies support the involvement of numerous genes in autistic disorders.About 1.7–4.8% of people with ASD have chromosome abnormalities. Almost all chromosomes have been involved, including unbalanced translocations, inversions, rings, and interstitial or terminal deletions and duplications.^11,12,13,14 The rare chromosome abnormalities that have been reported on more than one occasion are duplication of 15q,¹⁵ deletions of 18q,^16,17 Xp,^18,19 2q37,²⁰ 22q13^21,22 and the sex chromosome aneuploidies 47,XYY^23,24 and 45,X/46,XY.^25,26 This diversity of loci suggests that studying chromosomal aberrations in relationship to autism will require efficient and highly sensitive tools. In addition to the importance for diagnosis, identification of chromosomal imbalances in patients with ASD may also be instrumental for cloning disease‐causing genes. Analysis of Xp22.3 deletion has indeed allowed the identification of the NLGN4 gene.²⁷Recent technological developments, such as array‐based comparative genomic hybridisation (array‐CGH),^28,29,30 allow the investigation of the human genome at a resolution that is 5–10 times higher than that of routine chromosome analysis by karyotyping.^29,31,32,33 Array‐CGH has been used successfully for analysis of tumour samples and cell lines, and for high‐resolution analysis of patients with mental retardation and congenital anomalies.^{34,35,36,37,38}Here, we report the application of genomewide array‐CGH, at 1 Mb resolution, to the study of 29 patients with syndromic ASD. In addition to their clinical relevance, our results emphasise the importance of chromosomal imbalance in the aetiology of syndromic ASD and may help the identification of new genes involved in autistic disorders.     

Detection of BRAF V600E activating mutation in papillary thyroid carcinoma using PCR with allele-specific fluorescent probe melting curve analysis

Background

A single hotspot mutation at nucleotide 1799 of the BRAF gene has been identified as the most common genetic event in papillary thyroid carcinoma (PTC), with a prevalence of 29–83%.

Aims

To use a PCR assay to molecularly characterise the BRAF activating point mutation in a series of PTC and benign thyroid cases and correlate the mutation results with histological findings.

Methods

Formalin‐fixed paraffin‐embedded (FFPE) sections were evaluated for the BRAF V600E mutation using LightCycler PCR with allele‐specific fluorescent probe melting curve analysis (LCPCR).

Results

42 (37 PTC; 5 benign) surgical tissue samples were analysed for the BRAF V600E activating point mutation. Using LCPCR and direct DNA sequencing, the BRAF mutation was identified in 23/37 (62.2%) PTC FFPE samples. DNA sequencing results demonstrated confirmation of the mutation.

Conclusions

Detection of BRAF‐activating mutations in PTC suggests new approaches to management and treatment of this disease that may prove worthwhile. Identification of the BRAF V600E activating mutation in routine FFPE pathology samples by a rapid laboratory method such as LCPCR could have significant value.Although thyroid cancer represents only 1% of all human malignancies, it accounts for more than 90% of all endocrine cancers.¹ The incidence of thyroid cancer has risen in the United States, from a rate of 3.6 per 100 000 in 1973 to 8.7 per 100 000 in 2002.² The increase in thyroid cancer incidence can be attributed primarily to an increase in the incidence of papillary thyroid carcinoma (PTC). For the time period 1973 to 2002, the incidence of PTC increased from 2.7 to 7.7 per 100 000, a 2.9‐fold increase.² In a report of 15 700 patients in the United States, overall survival rates, corrected for age and sex, were 98% for PTC, 92% for follicular carcinoma, 80% for medullary carcinoma, and 13% for anaplastic carcinoma.³ Among the most curable of cancers, PTC tends to remain localised in the thyroid gland, but in time it may metastasise to regional lymph nodes and, less commonly, to the lungs. Peak incidence of PTC is in the fifth decade of life and it occurs nearly three times more frequently in women than in men.⁴A single hotspot mutation at nucleotide 1799 of the BRAF gene has been identified as the most common genetic event in PTC, with a prevalence of 29–83%.^{5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24} Activating BRAF mutations may be an important event in the development of PTC. This mutation had been formerly termed T1796A, based on the NCBI GenBank nucleotide sequence NM 004333, which missed a codon (three nucleotides) in exon 1 of the BRAF gene. With the correct version of the NCBI GenBank nucleotide sequence NT 007914 available, this BRAF mutation is now designated T1799A.²⁵ This thymine (T) to adenine (A) transversion mutation (T→A) results in the substitution of valine with glutamate in codon 600 (V600E, formerly V599E) and converts BRAF into a dominant transforming protein that causes constitutive activation of the mitogen‐activated protein kinase (MAPK) pathway, independent of RAS activation.²⁶ Amino acid 600 lies in the kinase domain of BRAF, and the V600E mutation makes the enzyme more active than wild‐type BRAF. The resulting protein shows increased kinase activity that can transform NIH3T3 cells.²⁶ This suggests that therapy with RAF kinase inhibitors may be of use in this disease. If the response to RAF kinase inhibition is dependent on the presence of an activated BRAF protein, it will be necessary to evaluate cases of PTC for the presence or absence of mutations.The BRAF V600E activating point mutation appears to be highly specific for PTC, with no benign or other well‐differentiated thyroid neoplasm having been found to harbour this mutation. Moreover, some studies have suggested that BRAF mutation may serve as a novel prognostic biomarker that predicts poor clinicopathological outcomes, helping to identify patients who should undergo more aggressive clinical follow‐up.^7,9,27 While these preliminary reports are somewhat controversial^{17,18,21,28,29,30} and additional studies are required to establish the efficacy of this marker, these findings suggest that BRAF mutation detection may serve as a useful tool for diagnosis, management and treatment of PTC.LightCycler PCR with allele specific fluorescent probe melting curve analysis (LCPCR) has been used successfully to detect BRAF activating point mutations in PTC.⁷ Recently, we reported on the clinical utility of this method for detecting BRAF mutations in a series of indeterminate thyroid fine needle aspirate (FNA) cytology samples.³¹ Based on the detection of either a fluorescent reporter probe or double‐stranded DNA‐binding dyes, “real‐time” PCR instruments, such as the LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) can provide quantitative information regarding target nucleic acid sequences. The LightCycler is a microvolume fluorimeter integrated with a thermal cycler. Using post‐amplification melting curve analysis, the LightCycler instrument can also be used to differentiate alleles for the purpose of determining sequence variations or point mutations. In this report, we used LCPCR to molecularly characterise the BRAF activating point mutation in a series of PTC and benign thyroid cases and correlate the mutation results with histological findings.     

Protein kinase C isoform expression as a predictor of disease outcome on endocrine therapy in breast cancer

Background

Although in vitro breast cancer models have demonstrated a role for protein kinase C (PKC) α and δ isoforms in endocrine insensitivity and resistance respectively, there is currently little clinical evidence to support these observations.

Aims

To define the pattern of PKC α and δ expression using breast cancer cell lines, with and without endocrine resistance, and also breast cancer samples, where expression can be correlated with clinicopathological and endocrine therapy outcome data.

Methods

PKC isoform expression was examined in tamoxifen responsive, oestrogen receptor positive (ER⁺), ER⁺ acquired tamoxifen resistant (TAM‐R) and oestrogen receptor negative (ER⁻) cell lines by western blotting and immunocytochemical analysis. PKC isoform expression was then examined by immunohistochemistry in archival breast cancer specimens from primary breast cancer patients with known clinical outcome in relation to endocrine response and survival on therapy.

Results

ER⁺ breast cancer cell lines expressed considerable PKC‐δ but barely detectable levels of PKC‐α, whereas ER⁻ cell lines expressed PKC‐α but little PKC‐δ. ER⁺ acquired TAM‐R cell lines expressed substantial levels of both PKC‐α and δ. In clinical samples, high PKC‐δ expression correlated to endocrine responsiveness whereas PKC‐α expression correlated to ER negativity. PKC‐δ was an independent predictor of duration of response to therapy. Patients showing a PKC‐δ⁺/PKC‐α⁻ phenotype had a six times longer endocrine response than patients with the PKC‐δ⁺/ PKC‐α⁺ phenotype (equating to tamoxifen resistance in vitro).

Conclusions

Levels of PKC‐α and δ expression appear to be indicative of response to anti‐oestrogen therapy and could be useful in predicting a patient''s suitability for endocrine therapy.Anti‐hormone therapies such as tamoxifen are widely used to treat breast cancer patients.¹ A small but significant number of patients receiving tamoxifen however will not respond or will develop resistance.^2,3 Many mechanisms have been suggested which may play a role in tamoxifen resistance but the mechanisms have not yet been fully elucidated.^4,5,6,7 Although rapid progress is being made in understanding the biology of oestrogen receptor (ER) function, the only predictive markers for endocrine therapy that currently yield sufficient levels of evidence to be recommended for routine practice, are ER and progesterone receptors, and to a lesser extent HER‐2 status.¹ Better ways of predicting which patients are suitable for endocrine therapy would prove useful in the fight against breast cancer. Expression of the signal transduction molecule, protein kinase C (PKC) is increased in breast cancer models of poor prognosis; for example, ER⁻ cell lines express significantly more PKC than ER⁺ cell lines.^8,9 However, multiple isoforms of PKC exist, with variation in their expression profile and mechanism of activation.^10,11,12 We have previously shown that ER⁺ MCF‐7 cell lines have high PKC‐δ and low PKC‐α expression, whereas ER⁻ MDA‐MB‐231 cells have high PKC‐α and low PKC‐δ expression. A wealth of literature now support these observations^13,14,15,16 linking PKC‐α expression to loss of ER expression and adverse cellular features,^13,15 and there is also emerging data that PKC‐δ expression can relate to loss of endocrine sensitivity in vitro.¹⁷ These studies did not however investigate the effect of PKC‐δ expression on clinical outcome. Moreover, a recent clinical study showed PKC‐α to be decreased in advanced breast cancer samples,¹⁸ suggesting that laboratory observations may not translate to the clinic.We have therefore established cell line models of tamoxifen resistance,^19,20 and used these models and well‐characterised clinical specimens^21,22 with known response to endocrine therapy, to study PKC expression. We have shown that PKC‐α and δ may prove useful in predicting whether patients will respond or not to endocrine therapy.     

Screening of calpain-3 autolytic activity in LGMD muscle: a functional map of CAPN3 gene mutations

Background

The diagnosis of calpainopathy is obtained by identifying calpain‐3 protein deficiency or CAPN3 gene mutations. However, in many patients with limb girdle muscular dystrophy type 2A (LGMD2A), the calpain‐3 protein quantity is normal because loss‐of‐function mutations cause its enzymatic inactivation. The identification of such patients is difficult unless a functional test suggests pursuing a search for mutations.

Materials and methods

A functional in vitro assay, which was able to test calpain‐3 autolytic function, was used to screen a large series of muscle biopsy specimens from patients with unclassified LGMD/hyperCKaemia who have previously shown normal calpain‐3 protein quantity.

Results

Of 148 muscle biopsy specimens tested,17 samples (11%) had lost normal autolytic function. CAPN3 gene mutations were identified in 15 of 17 patients (88%), who account for about 20% of the total patients with LGMD2A diagnosed in our series.

Conclusions

The loss of calpain‐3 autolytic activity is highly predictive of primary calpainopathy, and the use of this test as part of calpainopathy diagnosis would improve the rate of disease detection markedly. This study provides the first evidence of the pathogenetic effect of specific CAPN3 gene mutations on the corresponding protein function in LGMD2A muscle and offers new insights into the structural–functional relationship of the gene and protein regions that are crucial for the autolytic activity of calpain‐3.Limb girdle muscular dystrophies (LGMDs) comprise a clinically and genetically heterogeneous group of diseases usually characterised by progressive muscle weakness and wasting of pelvic and shoulder girdles. LGMD type 2A (LGMD2A, MIM 253600) was the first form of LGMD to be mapped and molecularly characterised, probably because it is the most frequent.^1,2,3,4,5 LGMD2A is caused by mutations in the CAPN3 gene (MIM 114240) that encodes for a non‐structural protein, the enzyme called calpain‐3.¹ Calpain‐3 is the muscle‐specific member of a family of Ca²⁺‐dependent proteases, which are supposed to play a part in many intracellular processes, including cell motility, apoptosis, differentiation and cell cycle regulation, by modulating the biological activity of their substrates through limited and strictly controlled proteolysis. Calpain‐3 is composed of four functional domains, and has three exclusive sequence inserts (NS, IS1 and IS2). The activation of calpain‐3 depends on phospholipids and Ca²⁺ ions,^6,7 takes place after unknown stimuli, and results in partial autolytic degradation.^8,9,10,11,12The molecular diagnosis of calpainopathy is complex because of the variability of clinical phenotypes, the effort required to identify point mutations in a relatively large gene, and incomplete sensitivity and specificity of calpain‐3 protein analysis on muscle biopsy specimen.^{13,14,15,16,17,18,19}An increasing number of studies have reported patients with LGMD2A whose diagnosis had been obtained by mutation identification despite normal levels of calpain‐3 protein in their muscle biopsy specimen.^4,15,20,21 As calpain‐3 is an enzyme, some mutations may not cause a reduction in protein content but its functional inactivation. Clinicians are increasingly aware that although the number of patients with LGMD2A showing normal calpain‐3 levels is marked, the identification of such cases is difficult. In patients with abnormal calpain‐3 protein levels, the effort required to search for mutations is justified because it will almost certainly confirm the LGMD2A diagnosis.¹⁶ Conversely, in patients with normal calpain‐3 protein content, an alternative diagnosis is usually pursued. For this reason, many patients with LGMD2A remain undiagnosed, unless a search is conducted for the CAPN3 gene mutation even if protein results are not indicative. To overcome this problem, we have developed a functional in vitro assay that is able to test the calpain‐3 autolytic activity in muscle samples,¹⁵ and showed that the loss of this function is associated with specific CAPN3 gene mutations.We report the results of extensive use of this functional assay as a screening tool in muscle biopsy specimens from patients with unclassified LGMDs and normal calpain‐3 protein quantity. Besides identifying patients with loss‐of‐function mutations, which would improve the rate of detection of the disease, we aimed at designing a functional map of the mutations and the protein regions involved in the autolytic activity to determine how single mutations exert their deleterious effect on the mutant protein.     

New VMD2 gene mutations identified in patients affected by Best vitelliform macular dystrophy

Purpose

The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin‐1 (hBest1) which is a Ca²⁺‐sensitive chloride channel. This study was performed to identify disease‐specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl⁻ channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease.

Methods

Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK‐293 cells and the associated Cl⁻ current was examined using whole‐cell patch clamp analysis.

Results

Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non‐functional. Furthermore, the Q293H mutant inhibited the function of wild‐type bestrophin‐1 channels in a dominant negative manner.

Conclusions

This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease‐linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane.Best disease, also called Best vitelliform macular dystrophy (BVMD), is a bilateral, progressive disease of the retinal pigment epithelium (RPE) leading to decreased visual acuity. Best disease has an autosomally dominant transmission, but the penetrance is incomplete and its expression is highly variable.^1,2,3 Although Best disease is the second most common form of juvenile macular degeneration, with an onset usually before 15 years of age, only about 1% of all cases of macular degeneration can be attributed to Best disease.⁴ However, the degree of central vision impairment and the age of onset of symptoms varies widely,^5,6,7 even among members of the same family.On fundus examination, the central macula has a transient “egg yolk”‐like appearance measuring between one and two disc areas in size. During angiographic examination, a blockade of the choroidal fluorescence by vitelliform material was observed.⁸ This means the lesion is an abnormal accumulation of lipofuscin‐like material in front of the choroid and within and beneath the RPE, but not within the neural retina. At the vitelliform stage, visual acuity is surprisingly good or slightly subnormal. The lesion evolves through several stages over many years (scrambled egg stage, cyst stage, pseudohypopyon stage, atrophic stage, see classification by Mohler and Fine⁹), with visual acuity usually decreasing when disintegration of the yellowish material has been observed (scrambled egg stage).⁸ When atrophic changes take place, visual acuity may drastically be reduced. Infrequently, in some patients, the lesion may degenerate, resulting in the development of subretinal haemorrhage with identifiable or unidentifiable choroidal neovascular membranes.^10,11,12,13 Some people without any subretinal macular deposit but who carry causative BVMD mutations may never experience a noticeable decline of central vision.About 10% of affected eyes have multifocal lesions in the extrafoveal region. Furthermore, lesions similar to those seen in Best disease may occur in patients with adult vitelliform macular dystrophy (AVMD).^14,15 In some cases, similar conditions are caused by mutations of other genes, such as the peripherin/RDS gene.¹⁶The differential diagnosis of BVMD from other macular dystrophies is most effectively made by measuring the electrooculogram (EOG),^17,18 whereas full‐field electroretinograms are usually normal in patients with BVMD.¹ Although for years the EOG has been considered as the main functional test to define BVMD and was used especially for the detection of non‐manifesting carriers of the mutated VMD2 gene, recent studies have reported normal EOG recordings in patients with BVMD.^6,7,19 Thus, it seems that a normal EOG alone may not unequivocally exclude non‐manifesting carriers. A complete clinical examination of patients combined with molecular genetics studies of the VMD2 gene is mandatory for adequate counselling of the families. In both affected patients and carrier patients, EOG often shows an abnormal light‐peak/dark‐trough ratio. Abnormal EOG responses can be recorded in asymptomatic patients, sometimes long before the appearance of any clinical manifestations. Multifocal electroretinography (mfERG) shows variable central function loss depending on the stage of the disease and has become an important tool in assessing the function of the remaining macular cones.^20,21The mutations responsible for Best disease are found in a gene called VMD2. It encodes a transmembrane protein named bestrophin‐1 (hBest1). The protein is located in the basolateral plasma membrane of RPE cells.²² Bestrophin is a member of the RFP‐TM family of proteins, so named for their highly conserved arginine, phenylalanine, proline (RFP) motif.^23,24,25 Bestrophin contains several domains that are highly conserved between species.²³ Patch clamp studies of bestrophin and other RFP‐TM family members heterologously overexpressed in cell culture have suggested that bestrophin is a Ca²⁺‐sensitive chloride channel.^26,27,28Some mutations in the VMD2 gene have also been associated with some cases of bull''s‐eye maculopathy²⁹ and of AVMD.^29,30,31,32In this study, we identify six new, independent, disease‐specific mutations in patients with BVMD and their families, and in isolated patients. One of these mutations (Q293H) found in a large family from the west part of France is particularly severe. Patch clamp analysis of human embryonic (HEK) cells expressing the Q293H mutant bestrophin‐1 shows that this mutant channel is non‐functional. Furthermore, the Q293H mutant inhibits the function of wild‐type bestrophin‐1 channels in a dominant negative manner. These findings support the idea that BVMD is a chloride channelopathy.     

Y-chromosome haplogroups and susceptibility to azoospermia factor c microdeletion in an Italian population

Background

A limited number of studies aimed at investigating the possible association of Y‐chromosome haplogroups with microdeletions of the azoospermia factors (AZFs) or with particular infertile phenotypes, but definitive conclusions have not been attained. The main confounding elements in these association studies are the small sample sizes and the lack of homogeneity in the geographical origin of studied populations, affecting, respectively, the statistical power and the haplogroup distribution.

Materials and methods

To assess whether some Y‐chromosome haplogroups are predisposing to, or protecting against, azoospermia factor c (AZFc; b2/b4) deletions, 31 north Italian patients carrying the AZFc b2/b4 microdeletion were characterised for 8 Y‐chromosome haplogroups, and compared with the haplogroup frequency shown by a north Italian population without the microdeletion (n = 93).

Results and discussion

A significant difference was observed between the two populations, patients with microdeletions showing a higher frequency of the E haplogroup (29.3% vs 9.7%, p<0.01). The geographical homogeneity of the microdeleted samples and of the control population, controlled at microgeographical level, allows the possibility that the geographical structure of the Y genetic variability has affected our results to be excluded.

Conclusion

Thus, it is concluded that in the north Italian population Y‐chromosome background affects the occurrence of AZFc b2/b4 deletions.Y‐chromosome long‐arm microdeletions are found in 5–10% of men with severe oligospermia and non‐obstructive azoospermia, and encompass one or more azoospermia factor (AZF) loci. Deletions of the azoospermia factor c (AZFc) region are clearly among the most commonly known molecular causes of spermatogenic failure in men.¹ These deletions are caused by homologous recombination between the 229‐kb‐long b2 and b4 amplicons² and span 3.5 Mb. Eight different gene families are removed by AZFc deletions, including all members of the DAZ gene family, which represents the stronger candidate for the AZFc phenotype.^{1,2,3,4,5,6,7} Although all AZFc deletions are essentially identical in molecular extension, people carrying these microdeletions present variable infertile phenotypes, suggesting the involvement of environmental factors and/or other genetic regions. Furthermore, the function of the AZF genes in human spermatogenesis and the role of the Y‐chromosome background in the predisposition to occurrence of deletions is still largely unknown.At present, around 250 Y single‐nucleotide polymorphisms have been discovered and their phylogenetic relationships are well known.⁸ These polymorphic markers of the male‐specific region of the Y chromosome define monophyletic groups of the Y chromosome, which hereafter we will name as “haplogroups”.A limited number of studies have investigated the possible association of Y‐chromosome haplogroups with microdeletions or with a particular infertile phenotype,⁹ but the contribution of predisposing factors or genetic background to causing deletions is still debated. In particular, only three studies have investigated the possible association between Y‐chromosome haplogroups and AZF deletions,^10,11,12 all of them failing to establish important associations. These works studied such associations in an European population involving 73 microdeleted samples of heterogeneous geographical origin,¹⁰ in a northwestern European population involving 50 patients¹¹ and in a Japanese population, more geographically localised but represented by a very low number of people with microdeletions (six patients).¹² All the previous studies that found some suggestion of an association with Y‐chromosome haplogroups dealt with infertility. They reported a considerable over‐representation of the haplogroup K(xL,N,O1,O3c,P) in Danish men with reduced sperm count, which did not reach significance probably because of a small sample size,¹³ and D2b Y lineage in Japanese men with reduced sperm count,¹⁴ not confirmed by a later study.¹²However, these association studies require particular attention to two principal factors: (1) the geographical structure of the Y‐chromosome variations in the population under investigation, because the Y‐chromosome genetic variability is highly geographically structured and the Y‐haplogroup distribution changes over different geographical areas¹⁵; and (2) the number and selection criteria of the patient and control groups.To assess whether some Y‐chromosome haplogroups predispose to, or protect against, AZFc deletion, we have defined and compared Y‐chromosome haplogroup distribution in a group of unrelated Italian infertile men harbouring the b2/b4 deletion (n = 41, 31 of whom were from north Italy) and in a control group represented by fertile men without microdeletions (fathers of at least one child) from north Italy (n = 93).     

A comprehensive strategy for the subtyping of patients with Fanconi anaemia: conclusions from the Spanish Fanconi Anemia Research Network

Background

Fanconi anaemia is a heterogeneous genetic disease, where 12 complementation groups have been already described. Identifying the complementation group in patients with Fanconi anaemia constitutes a direct procedure to confirm the diagnosis of the disease and is required for the recruitment of these patients in gene therapy trials.

Objective

To determine the subtype of Fanconi anaemia patients in Spain, a Mediterranean country with a relatively high population (23%) of Fanconi anaemia patients belonging to the gypsy race.

Methods

Most patients could be subtyped by retroviral complementation approaches in peripheral blood T cells, although some mosaic patients were subtyped in cultured skin fibroblasts. Other approaches, mainly based on western blot analysis and generation of nuclear RAD51 and FANCJ foci, were required for the subtyping of a minor number of patients.

Results and conclusions

From a total of 125 patients included in the Registry of Fanconi Anaemia, samples from 102 patients were available for subtyping analyses. In 89 cases the subtype could be determined and in 8 cases exclusions of common complementation groups were made. Compared with other international studies, a skewed distribution of complementation groups was observed in Spain, where 80% of the families belonged to the Fanconi anaemia group A (FA‐A) complementation group. The high proportion of gypsy patients, all of them FA‐A, and the absence of patients with FA‐C account for this characteristic distribution of complementation groups.Fanconi anaemia is a rare hereditary recessive disease characterised by developmental abnormalities, bone marrow failure and predisposition to cancer, mainly acute myeloid leukaemia.¹ To date, 12 complementation groups have been reported (FA‐A, B, C, D1, D2, E, F, G, I, J, L and M) and 11 associated genes have already been identified: FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, BRIP1/FANCJ, FANCL and FANCM/Hef.^{2,3,4,5,6,7,8,9,10,11,12,13,14} In dividing cells or in cells exposed to DNA damage, eight Fanconi anaemia proteins (FANCA/B/C/E/F/G/L/M) form a Fanconi anaemia core complex, necessary for the monoubiquitination of FANCD2.^2,5,15 In contrast with these Fanconi anaemia proteins, FANCD1 and FANCJ are not involved in FANCD2 monoubiquitination, indicating that these proteins participate downstream of FANCD2 in the FA/BRCA pathway.^2,7Because of the overlap in the phenotype and molecular pathways between the different chromosome fragility syndromes, Fanconi anaemia subtyping constitutes an invaluable approach to confirm the diagnosis of the disease.^16,17 Additionally, in the case of patients with FAD1, subtyping analysis allows the identification of BRCA2 mutation carriers, characterised by an increased risk of developing breast, ovarian and other types of cancers.¹⁸ Fanconi anaemia subtyping also facilitates mutation screening studies and therefore the identification of mutations with particular pathogenic effects. In addition to the above‐mentioned applications, subtyping is essential before enrolling a patient with Fanconi anaemia in a gene therapy trial.Progress in the cloning of Fanconi anaemia genes enabled the identification of mutations in specific Fanconi anaemia genes by means of DNA sequencing approaches or other methods.¹⁹ The large number and complexity of some Fanconi anaemia genes and their mutations, together with the necessity of verifying the pathogenicity of each new mutation, implies that subtyping of patients with Fanconi anaemia by mutational analysis is often time consuming and laborious. The possibility of reverting the phenotype of Fanconi anaemia cells by the transfer of functional Fanconi anaemia genes has been recently proposed as an efficient approach for identifying the pathogenic genes that account for the disease in patients with Fanconi anaemia.^20,21 A different Fanconi anaemia subtyping approach is based on the western blot analysis of FANCD2.²² By means of the observation of the ubiquitinated (FANCD2‐L) and non‐ubiquitinated (FANCD2‐S) forms of the protein FANCD2, it is possible to predict pathogenic mutations in proteins upstream or downstream of FANCD2.²³ In the case of patients belonging to rare complementation groups such as FAD1 or FA‐J, approaches based on the formation of RAD51 or BRIP1 nuclear foci are also highly informative in identifying their complementation group.²⁴With the purpose of determining the prevalence of the different Fanconi anaemia complementation groups in Spain, we conducted an extensive subtyping study of Fanconi anaemia in this Mediterranean country. In addition to a predominantly caucasian population, a relatively large population of about 500 000 gypsies also live in Spain. In this population, the incidence of recessive syndromes is high, owing to the high rates of consanguinity.²⁵ This study will allow us to identify potential differences in the distribution of Fanconi anaemia subtypes due to geographical and ethnic characteristics, and will also allow us to conduct further mutation studies within the population of patients subtyped for Fanconi anaemia. Additionally, our subtyping study will facilitate the enrolling of patients with Fanconi anaemia in clinical gene therapy trials aimed at the genetic correction of their haematopoietic stem cells.     

Mucosal endocrine cell micronests and single endocrine cells following neo-adjuvant therapy for adenocarcinoma of the distal oesophagus and oesophagogastric junction

Aims

To determine the frequency of endocrine cell micronests (ECM) and single endocrine cells (SEC) within the glandular mucosa of the distal oesophagus and oesophagogastric junction (OGJ) following neo‐adjuvant therapy for adenocarcinoma.

Methods

The resection specimens from 11 patients with adenocarcinoma of the distal oesophagus or OGJ who had undergone preoperative chemotherapy or chemoradiotherapy (CRT) were reviewed and stained immunohistochemically for cytokeratin and chromogranin. The presence of ECM and/or SEC within the mucosa adjacent to the tumour was noted, and the results correlated with the extent of tumour regression. The corresponding pretreatment endoscopic biopsy specimens were reviewed in 6 cases, and the results were also compared to 10 tumour resections from patients with no history of neo‐adjuvant treatment.

Results

ECM and/or SEC were identified in 8/11 resection specimens after chemotherapy or CRT. The endocrine cells were typically located within the deep lamina propria or muscularis mucosae and were associated with varying degrees of glandular atrophy and inflammation. The appearances were most consistent with endocrine cell preservation (pseudo‐hyperplasia) following treatment. Isolated endocrine elements were not seen in the pretreatment biopsy specimens, while rare SEC without ECM were identified in only 2/10 control resection specimens.

Conclusions

Endocrine cell pseudo‐hyperplasia may be seen within atrophic glandular mucosa following neo‐adjuvant therapy of distal oesophageal/OGJ adenocarcinomas. The changes are analogous to those seen in chronic atrophic gastritis and should not be misinterpreted as those of residual tumour.Adenocarcinoma of the distal oesophagus and the oesophagogastric junction (OGJ) is now the most common type of oesophageal cancer and appears to be increasing in incidence.^1,2,3 Although surveillance of patients with Barrett''s oesophagus has led to early diagnosis in some cases, many patients still present with relatively advanced disease and the overall prognosis is poor. The management of patients with potentially curable disease is dependent on the tumour stage and while superficial tumours may be amenable to local ablation, most patients require distal oesophagectomy and proximal gastrectomy. Recently, chemotherapy and/or radiotherapy (CRT) have been introduced prior to surgery with the intention of improving loco‐regional control by reducing tumour volume and eradicating nodal metastases (“down‐staging”). While there is no clear evidence that neo‐adjuvant CRT improves overall survival,^1,2 patients with complete pathological remission at the time of resection appear to have a better prognosis.^3,4,5,6,7The accurate assessment of tumour staging and the identification of any treatment‐related effects depend on the detailed histological examination of resection specimens. Therefore it is self‐evident that pathologists need to be aware of the morphological changes that may occur in the oesophagus or stomach following neo‐adjuvant therapy. Complete microscopic regression of tumour has been documented in 10–30% cases, while the remaining specimens show variable degrees of persistent neoplasia.^2,5 Characteristic treatment induced changes within the residual tumour include attenuation of glands, increased cytological atypia, degenerative nuclear changes and decreased mitotic activity.⁸ Cytoplasmic vacuolation and eosinophilia have been described in rectal adenocarcinomas after therapy, and the latter appearances may reflect an endocrine or oncocytic phenotype.^9,10 Stromal changes that commonly follow treatment include fibrosis, dystrophic calcification, atypical fibroblasts and vascular obliteration. Mucin pools that are acellular or include only scant residual neoplastic cells are reported in 11% of Barrett''s oesophagus‐related adenocarcinomas, and this feature is more common in tumours with mucinous or signet ring cell morphology prior to therapy.¹¹ The relative proportions of the reactive fibro‐inflammatory stroma and the residual neoplastic elements have been used to grade the effects of CRT in oesophageal adenocarcinoma,^5,7,8,12 and similar grading systems have been applied in tumours of the stomach¹³ and rectum.^14,15,16 Recently, one such grading scheme was shown to have excellent inter‐observer agreement.¹⁷While changes in the neoplastic cells and the stroma following treatment are relatively well documented, few studies have detailed the appearance of the non‐neoplastic mucosa other than the documentation of associated ulceration, Barrett''s oesophagus or dysplasia. However focal glandular atrophy, epithelial attenuation and apoptosis have been described within the specialised gastric mucosa,⁸ and Brien and colleagues noted that radiation induced atypia within benign glands can mimic dysplasia or even residual carcinoma.¹⁸ Similar epithelial changes together with crypt architectural distortion and fibrosis within the lamina propria and submucosa have been described in treated rectal tumours.^19,20 We recently observed prominent clusters of endocrine cells (endocrine cell micronests, ECM) and isolated endocrine cells within the non‐neoplastic mucosa adjacent to areas of ulceration and tumour regression in oesophagogastric resection specimens following neo‐adjuvant therapy. Since, to our knowledge, ECM have not been described in this clinical setting, we examined a series of resection specimens from patients who had undergone preoperative chemotherapy or CRT and compared the results with corresponding preoperative biopsy specimens, and with resection specimens from control cases in which there was no history of prior treatment.