Molecular diagnosis of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Its estimated prevalence is 1 per 800 individuals. ADPKD patients constitute 8% of the population on dialysis or kidney transplantation. The disease can be diagnosed using radiological or genetic procedures. Direct genetic diagnosis of the disease can now be performed in Spain; however, it is not an easy or cheap test. This is why every case should be considered individually to determine whether genetic testing is appropriate, and to determine which genetic test is most adequate. Genetic testing in ADPKD is of special interest for living donors and neonatal and sporadic cases. Genetic testing offers the chance of performing prenatal or pre-implantation testing of embryos in families with severe cases of the disease. Also, this will enable the disease to be treated, when specific treatment becomes available, in cases that would not be candidates for treatment without genetic confirmation.     

Molecular analysis of autosomal dominant neurohypophyseal diabetes insipidus

The status of the arginine vasopressin-neurophysin-II (AVP-NPII) gene was studied in three families with autosomal dominant neurohypophyseal diabetes insipidus (AD-NDI). Restriction fragments of genomic DNA containing AVP-NPII sequences from affected individuals were not detectably different in size from those of normal controls. Thus, these individuals with ADNDI do not have apparent large deletions, insertions, or rearrangements of an AVP-NPII allele. Four restriction fragment length polymorphisms were detected with a probe for the adjacent gene on chromosome 20, oxytocin-neurophysin-I (OT-NPI). Linkage studies in these three families between the restriction fragment length polymorphism haplotypes and ADNDI phenotype strongly suggest cosegregation. This indicates that the genetic locus for ADNDI maps within or near the AVP-NPII locus and suggests that a defective AVP-NPII allele may be the basis of ADNDI.     

Detection of familial hypercholesterolemia in a cohort of children with hypercholesterolemia: results of a family and DNA-based screening

The diagnosis of familial hypercholesterolemia (FH) in unselected children is difficult due to the frequent overlap of cholesterol values in affected and non-affected and the paucity of physical signs. Nevertheless, detection and treatment of FH in childhood has been advocated to prevent atherosclerosis in these patients. Here, we report the results of a screening program in a cohort of 157 unrelated, hypercholesterolemic (HC) children (age range 2-15 years; mean 8.3+/-3.4 years) carried out by a combination of family study and molecular analysis of the LDLR gene. On the basis of the familial phenotype, 27 (17.2%) were classified as probable FH and 49 (31.2%) as affected by FCHL. Among probable FH children, 14 (51.8%) carried mutant LDLR alleles, giving an overall 8.9% prevalence of FH. Most of LDLR variants were already reported, but three new mutations G266C, T368M, and D451Y were identified. Beside increased TC and LDL-C (p<0.001), FH children showed decreased HDL-C (p<0.05) and higher prevalence of family history of CAD when compared to non-FH children. None presented tendon xanthomas. We estimated that LDL-C >3.9 mmol/L was the best cut off value for diagnosing FH in these children, showing 79% sensitivity and 71.0% specificity. We propose the use of a LDL-C cut off level associated with a family study to identify FH among HC children.     

Primary shunt hyperbilirubinaemia in a large four-generation family confirming autosomal dominant genetic disorder

AIM: To describe the pattern of inheritance and confirm the diagnostic criteria of primary shunt hyperbilirubinaemia (PSH). METHODS: Forty members of a family pedigree across four generations were included in this study. All family members were interviewed and investigated by physical examination, hematology and liver function test and the pattern of inheritance was analyzed. RESULTS: Nine of the forty family members suffered primary shunt hyperbilirubinaemia. The mature erythrocytes of the propositus were irregular in shape and size. The pedigree showed transmission of the trait through four generations with equal distribution in male and female. No individual with a primary shunt hyperbilirubinaemia was born to unaffected parents. The penetrance was complete in adult. CONCLUSION: The pattern of inheritance is autosomal dominant. The abnormality of erythrocytes and decrease in white blood cell could be supplemented in the diagnosis of PSH. The PSH is a genetic disorder and could by renamed as hereditary shunt hyperbilirubinaemia.     

Reduced penetrance of autosomal dominant hypercholesterolemia in a high percentage of families: importance of genetic testing in the entire family

Background

Autosomal dominant hypercholesterolemias (ADHs) are characterised by increased plasma levels of total and LDL cholesterol, predisposing to premature atherosclerosis. ADHs comprise several diseases with undistinguishable phenotype, caused by mutations in different genes: LDLR, APOB and PCSK9. Genetic studies are usually performed in patients with altered cholesterol levels. However, some persons carrying pathogenic mutations are normocholesterolemic and there are no further studies about this subject. We have studied the frequency of families and individuals carrying ADH mutations who do not present the disease in Spanish population.

Methods

We have analysed genes known to cause ADH by direct sequencing in 24 ADH families (215 members). Functional effect of some LDLR gene mutations was assessed by transfecting cultured cells with plasmids.

Results

Six families with mutations presented 7 mutation carriers who did not show ADH phenotype: 30% of ADH families presented normocholesterolemic individuals, and 7% of carriers of pathogenic mutations did not show ADH phenotype. We have analysed the effect of some of these mutations and they are responsible for impaired LDL receptor function. We have excluded mutations in APOB and PCSK9 genes that could reduce LDLc levels.

Conclusions

An important percentage of ADH families presented individuals who do not show an ADH phenotype, but who are able to transmit the pathogenic mutation to their offspring. Genetic study of all subjects in ADH families should be performed in order to identify normocholesterolemic carriers that allow the detection of mutations in their descendants and the prevention of the disease consequences.     

Characterization of a novel autosomal dominant bleeding disorder in a large kindred from east Texas

A large east Texas family with autosomal dominant inheritance of a novel bleeding disorder has been identified. The disorder is characterized clinically by easy bruising, life-threatening bleeding with trauma or surgery, and menorrhagia in affected women. Laboratory studies demonstrated prolongation of the prothrombin time and activated partial thromboplastin time in affected individuals. Paradoxically, assays of known coagulation factors are all within normal limits. To determine the molecular basis of this disease, a candidate gene linkage analysis in this kindred was done. Initially it was hypothesized that the cause of the disease in this family could be an antithrombin III (AT3) mutation that resulted in a constitutively active AT3 in the absence of heparin binding. Linkage studies using DNA from the family and an intragenic polymorphic marker within the AT3 gene showed that the disease mapped to this locus. The coding region and intron/exon junctions of AT3 were sequenced using the proband's DNA, but this analysis failed to identify a mutation. Additional family members were recruited for the study, and 16 polymorphic markers around the AT3 gene were analyzed. Using 2 recombinants, the critical interval for the defective gene was narrowed to approximately 1.5 Mb, centromeric to AT3. The factor V (FV) gene was mapped into the disease interval and sequenced; there were no mutations found. Elucidation of the genetic defect causing the bleeding disorder in this family may reveal a novel protein involved in the coagulation cascade.     

Familial hiatal hernia in a large five generation family confirming true autosomal dominant inheritance

BACKGROUND: Familial hiatal hernia has only rarely been documented. AIMS: To describe the pattern of inheritance of familial hiatal hernia within an affected family. SUBJECTS: Thirty eight members of a family pedigree across five generations. METHODS: All family members were interviewed and investigated by barium meal for evidence of a hiatal hernia. RESULTS: Twenty three of 38 family members had radiological evidence of a hiatal hernia. No individual with a hiatal hernia was born to unaffected parents. In one case direct male to male transmission was shown. CONCLUSIONS: Familial inheritance of hiatal hernia does occur. Evidence of direct male to male transmission points to an autosomal dominant mode of inheritance.     

Effects of ezetimibe coadministered with simvastatin on C-reactive protein in a large cohort of hypercholesterolemic patients

OBJECTIVE: This study assessed the effect of coadministration of ezetimibe and simvastatin on high sensitivity C-reactive protein (hs-CRP) in a large subject cohort (N=1089). METHODS: Data were combined from two nearly identical prospective trials. After dietary stabilization, washout period, and placebo lead-in period, patients with baseline low-density lipoprotein cholesterol (LDL-C) > or =3.75-6.50 mmol/l and triglycerides (TG) < or =4.0 mmol/l were randomized to one of the following daily treatments for 12 weeks: ezetimibe 10 mg; simvastatin monotherapy (10, 20, 40, or 80 mg); ezetimibe 10mg plus simvastatin (10, 20, 40, or 80 mg); or placebo. The primary analysis was the percent change in hs-CRP for the pooled ezetimibe plus simvastatin versus simvastatin monotherapy cohorts. RESULTS: Ezetimibe coadministered with simvastatin more than doubled the hs-CRP reduction compared to simvastatin monotherapy (-33.3% versus -14.3%, p<0.01). At each individual simvastatin dose level, coadministration therapy exerted significant further incremental hs-CRP reductions compared to simvastatin monotherapy. Similar hs-CRP reductions with coadministered ezetimibe and simvastatin were observed in the major subgroups examined (coronary heart disease, gender, age, baseline LDL-C, and body mass index). CONCLUSION: In this large subject cohort, ezetimibe coadministered with simvastatin significantly reduced hs-CRP, suggesting a possible additional anti-inflammatory/anti-atherosclerotic action of combination therapy compared to simvastatin monotherapy.     

Identification and characterization of new gain-of-function mutations in the PCSK9 gene responsible for autosomal dominant hypercholesterolemia

BackgroundThe identification of mutations in PCSK9 (proprotein convertase subtilisin kexin9) in autosomal dominant hypercholesterolemia (ADH), has revealed the existence of a new player in cholesterol homeostasis. PCSK9 has been shown to enhance the degradation of the LDL receptor (LDLR) at the cell surface. Gain-of-function mutations of PCSK9 induce ADH and are very rare, but their identification is crucial in studying PCSK9's role in hypercholesterolemia, its detailed trafficking pathway and its impact on the LDLR.MethodsIn order to identify new mutations and understand the exact mechanisms of action of mutated PCSK9, PCSK9 was sequenced in 75 ADH patients with no mutations in the LDLR or APOB genes. Functional analyses in cell culture were conducted and the impact of novel PCSK9 mutations on the quantitative and qualitative features of lipoprotein particles and on the HDL-mediated cellular cholesterol efflux was studied.ResultsAmong these 75 ADH probands with no mutations in the LDLR or APOB genes, four gain-of-function mutations of PCSK9 were identified, of which two were novel: the p.Leu108Arg and the p.Asp35Tyr substitutions. In vitro studies of their consequences on the activity of PCSK9 on cell surface levels of LDLR showed that the p.Leu108Arg mutation clearly results in a gain-of-function, while the p.Asp35Tyr mutation created a novel Tyr-sulfation site, which may enhance the intracellular activity of PCSK9.ConclusionThese data further contribute to the characterization of PCSK9 mutations and to better understanding of the impact on cholesterol metabolism of this new therapeutic target.     

After the LDL receptor and apolipoprotein B, autosomal dominant hypercholesterolemia reveals its third protagonist: PCSK9

The genes encoding the low-density lipoproteins receptor and its ligand apolipoprotein B, have been the only two genes classically implicated in autosomal dominant hypercholesterolemia. We have identified in 2003, the third gene implicated in this disease: PCSK9 (Proprotein Convertase Subtilin Kexin 9). Several mutations (p.S127R, p.F216L, p.D374Y...) of this gene have been reported to cause hypercholesterolemia by a gain of function leading to a reduction of LDL receptor levels. Other variations of PCSK9 are conversely associated with hypocholesterolemia particularly the non-sense p.Y142X and p.C679X mutations found in 2% of black Americans and associated with a decrease of LDL levels and coronary heart diseases. PCSK9 substrates and exact role have not been elucidated yet, but it seems that PCSK9 is definitely a major actor in cholesterol homeostasis. PCSK9 inhibitors might constitute new therapeutic targets that would decrease plasma LDL cholesterol levels and be synergistic with statin drugs.     

Relationship between caffeine intake and autosomal dominant polycystic kidney disease progression: a retrospective analysis using the CRISP cohort

Background

Caffeine has been proposed, based on in vitro cultured cell studies, to accelerate progression of autosomal dominant polycystic kidney disease (ADPKD) by increasing kidney size. Since ADPKD patients are advised to minimize caffeine intake, we investigated the effect of caffeine on disease progression in the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP), a prospective, observational cohort study.

Methods

Our study included 239 patients (mean age?=?32.3?±?8.9 ys; 188 caffeine consumers) with a median follow-up time of 12.5?years. Caffeine intake reported at baseline was dichotomized (any vs. none). Linear mixed models, unadjusted and adjusted for age, race, sex, BMI, smoking, hypertension, genetics and time, were used to model height-adjusted total kidney volume (htTKV) and iothalamate clearance (mGFR). Cox proportional hazards models and Kaplan-Meier plots examined the effect of caffeine on time to ESRD or death.

Results

Caffeine-by-time was statistically significant when modeling ln(htTKV) in unadjusted and adjusted models (p?<? 0.01) indicating that caffeine consumers had slightly faster kidney growth (by 0.6% per year), but htTKV remained smaller from baseline throughout the study. Caffeine consumption was not associated with a difference in mGFR, or in the time to ESRD or death (p?>?0.05). Moreover the results were similar when outcomes were modeled as a function of caffeine dose.

Conclusion

We conclude that caffeine does not have a significant detrimental effect on disease progression in ADPKD.

     

Cardiac magnetic resonance assessment of left ventricular mass in autosomal dominant polycystic kidney disease

Summary

Background and objectives

Autosomal dominant polycystic kidney disease (ADPKD) is associated with a substantial cardiovascular disease burden including early onset hypertension, intracranial aneurysms, and left ventricular hypertrophy (LVH). A 41% prevalence of LVH has been reported in ADPKD, using echocardiographic assessment of LV mass (LVM). The HALT PKD study was designed to assess the effect of intensive angiotensin blockade on progression of total kidney volume and LVM. Measurements of LVM were performed using cardiac magnetic resonance (MR).

Design, setting, participants, & measurements

Five hundred forty-three hypertensive patients with GFR >60 ml/min per 1.73 m² underwent MR assessment of LVM at baseline. LVM was adjusted for body surface area and expressed as LVM index (LVMI; g/m²).

Results

Baseline BP was 125.1 ± 14.5/79.3 ± 11.6 mmHg. Average duration of hypertension was 5.79 years. Prior use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers was present in 59.5% of patients. The prevalence of LVH assessed using nonindexed LVM (g) was 3.9% (n = 21, eight men and 13 women) and 0.93% (n = 5, one man and four women) using LVMI (g/m²). In exploratory analyses, the prevalence of LVH using LVM indexed to H^2.7, and the allometric index ppLVmass_HW, ranged from 0.74% to 2.23% (n = 4 to 12). Multivariate regression showed significant direct associations of LVMI with systolic BP, serum creatinine, and albuminuria; significant inverse associations with LVMI were found with age and female gender.

Conclusions

The prevalence of LVH in hypertensive ADPKD patients <50 years of age with short duration of hypertension, and prior use of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers is low. Early BP intervention in ADPKD may have decreased LVH and may potentially decrease cardiovascular mortality.     

Glomerular hyperfiltration and renal progression in children with autosomal dominant polycystic kidney disease

Summary

Background and objectives

The purpose of this study was to determine whether glomerular hyperfiltration (GH) occurring early in autosomal dominant polycystic kidney disease (ADPKD) is indicative of more rapid disease progression in children.

Design, setting, participants, & measurements

One hundred eighty children with ADPKD (ages 4 to 18 years) with normal renal function were examined by renal ultrasound. Renal volume was calculated using a standard formula for a modified ellipsoid. Creatinine clearance was calculated from serum creatinine and 24-hour urine creatinine. GH was defined as creatinine clearance ≥140 ml/min per 1.73 m².

Results

Thirty-two children had GH (mean age 11.4 ± 3.6 years) and 148 had normal renal function (mean age 10.8 ± 3.9 years). Patients with GH at baseline demonstrated an increased rate of total renal volume growth (β: rate of change = +19.3 ± 10.8 cm³/year) over 5 years compared with those without GH at baseline (β = −4.3 ± 7.7 cm³/year), P = 0.008. Those with GH at baseline experienced a faster decline in creatinine clearance in subsequent years (β = −5.0 ± 0.8 ml/min per 1.73 m² per year) compared with those without GH at baseline (β = +1.0 ± 0.4 ml/min per 1.73 m² per year), P < 0.0001.

Conclusions

This study revealed that occurrence of GH in ADPKD children is associated with a significantly faster decline in renal function and higher rate of kidney enlargement over time. GH combined with the increased renal volume may therefore be used as an early marker for a more severe progression of ADPKD in children.     

The White platelet syndrome: a new autosomal dominant platelet disorder

The present study has evaluated 46 members from four generations of a large family with mild to moderate bleeding symptoms, prolonged bleeding times and poor responses to all aggregating agents. Twenty-one members of the family had all of the abnormal structural features of the disorder brought to Minnesota in 1870 by Esther White and, therefore, referred to as the White platelet syndrome (WPS). Their platelet counts were decreased and mean platelet volumes increased. Four to 13% of their platelets contained large, fully developed Golgi complexes actively budding smooth and coated vesicles and frequently associated with centrioles. Such structures are usually present in megakaryocytes only during the major phase of granulopoiesis. As many as seven Golgi complexes and five centrioles were present in single platelets. Alpha granule formation appeared incomplete in patient platelets. The organelles were often immature in appearance and markedly decreased in number in many of their cells. As a result 30% or more of WPS platelets were "gray platelets" similar to, yet very different from the cells in patients with the gray platelet syndrome. Other abnormal features included cytoplasmic sequestration by residual dense tubular system membranes, autodigestion, larger than normal mitochondria and half normal-sized dense bodies. Thirteen of the 46 family members had a "touch" of WPS. Three to 5% of their platelets contained Golgi complexes. However, gray platelets were not present among their circulating cells. The WPS is a unique autosomal dominant condition that can be classified among the platelet granule deficiency disorders.     

Autosomal recessive form of isolated growth hormone deficiency is more frequent than the autosomal dominant form in a Brazilian cohort

BackgroundIn most studies, the autosomal dominant (type II) form of isolated growth hormone deficiency (IGHD) has been more frequent than the autosomal recessive (type I) form.Our aim was to assess defects in the GH1 in short Brazilian children with different GH secretion status.Subjects and methodsWe selected 135 children with postnatal short stature and classified according to the highest GH peak at stimulation tests in: severe IGHD (peak GH ≤ 3.3 μg/L, n = 38, all with normal pituitary magnetic resonance imaging); GH peak between 3.3 and 10 μg/L (n = 76); and GH peak > 10 μg/L (n = 21). The entire coding region of GH1 was sequenced and complete GH1 deletions were assessed by Multiplex Ligation Dependent Probe Amplification and restriction enzyme digestion.ResultsPatients with severe IGHD had a higher frequency of consanguinity, were shorter, had lower levels of IGF-1 and IGFBP-3, and despite treatment with lower GH doses had a greater growth response than patients with GH peak ≥ 3.3 μg/L. Mutations were found only in patients with severe IGHD (GH peak < 3.3 μg/L). Eight patients had autosomal recessive IGHD: Seven patients were homozygous for GH1 deletions and one patient was compound heterozygous for a GH1 deletion and the novel c.171 + 5G > C point mutation in intron 2, predicted to abolish the donor splice site. Only one patient, who was heterozygous for the c.291 + 1G > T mutation located at the universal donor splice site of intron 3 and predicts exon 3 skipping, had an autosomal dominant form.ConclusionAnalysis of GH1 in a cohort of Brazilian patients revealed that the autosomal recessive form of IGHD was more common than the dominant one, and both were found only in severe IGHD.     

Increased intestinal cholesterol absorption in autosomal dominant hypercholesterolemia and no mutations in the low-density lipoprotein receptor or apolipoprotein B genes

CONTEXT: Autosomal dominant hypercholesterolemia (ADH) is frequently caused by functional mutations in the low-density lipoprotein receptor (LDLR) or apolipoprotein B-100 (APOB) genes, but approximately 40% of ADH subjects disclose no such molecular defects, possibly pointing to alternative genetic mechanisms. OBJECTIVE: Our objective was to test the hypothesis that increased intestinal cholesterol absorption might play a role in the lipid abnormalities of subjects with ADH without identified genetic defects. DESIGN AND SETTING: This is a cross-sectional study of consecutive subjects with primary hyperlipidemia identified during an 18-month period in two lipid clinics. STUDY SUBJECTS: A total of 52 subjects with a clinical diagnosis of ADH were examined for molecular defects in LDLR and APOB. No APOB defects were found. Functional LDLR mutations occurred in 31 (60%) subjects, who received a diagnosis of familial hypercholesterolemia (FH). Those for whom no mutations could be identified were labeled as non-FH ADH. In addition, 38 subjects with familial combined hyperlipidemia (FCH) and 45 normolipidemic control subjects were studied. INTERVENTIONS: Interventions were diagnostic. MAIN OUTCOME MEASURES: Serum noncholesterol sterols were used as markers for the efficiency of intestinal cholesterol absorption. RESULTS: Adjusted campesterol to cholesterol ratios increased in the order non-FH ADH more than FH more than controls more than FCH, with mean values (95% confidence interval) in 10(2) mmol/mol cholesterol of 505 (424-600), 397 (345-458), 335 (294-382), and 284 (247-328), respectively. Thus, cholesterol absorption was lowest in FCH and highest in non-FH ADH. CONCLUSIONS: Increased intestinal cholesterol absorption may partially explain the high cholesterol levels of non-FH ADH subjects. Serum noncholesterol sterols are a useful tool for the differential diagnosis of genetic hypercholesterolemias, especially FCH and ADH unrelated to LDLR or APOB defects.     

Genetic thrombocytopenia with autosomal dominant transmission: a review of 54 cases

On the basis of a retrospective study of 3600 platelet kinetic studies, we have isolated 54 cases with chronic thrombocytopenia, a normal autologous and homologous platelet lifespan, and increased mean platelet volume without Döhle bodies, the absence of any functional platelet abnormalities, and a normal megakaryocyte count. These cases were either discovered during the first few years of life (i.e. constitutional) and/or were proven to be familial (autosomal dominant transmission). Previous treatments (corti-costeroids, immunoglobulins, androgens, immunosuppres-sor agents, splenectomy) were not effective in any of these cases or in their relatives. A new syndrome can therefore be proposed which can easily be suspected on the basis of platelet kinetic studies performed in cases of early onset, increased platelet volume, failure of corticosteroids or evidence of a familial blood disorder. It can be proved when the autologous platelet life span is demonstrated to be normal in spite of a chronic thrombocytopenia and a normal megakaryocytic count. The recognition of this syndrome will avoid neonatal complications (cephal-haematomas), surgical complications, and the use of expensive and possibly harmful ineffective treatments, both in the propositus and in other abnormal family members. The syndrome is certainly frequent (54 cases are presented here), but the diagnosis is often missed or delayed due to the low risk of haemorrhage. However, it is associated with a certain risk of leukaemia (four cases in three families).