Implications of Consanguinity for Routine Diagnostic Testing and Development of Specialist Services Teresa Lamb Clinical Scientist Leeds DNA Laboratory

Implications of Implications of Consanguinity for Consanguinity for

Routine Diagnostic Routine Diagnostic Testing and Testing and

Development of Development of Specialist ServicesSpecialist Services

Teresa LambTeresa Lamb

Clinical ScientistClinical Scientist

Leeds DNA LaboratoryLeeds DNA Laboratory

OutlineOutline

Relevance of consanguinity to Relevance of consanguinity to diagnostic molecular genetic diagnostic molecular genetic laboratorieslaboratories

Routine testingRoutine testing Autosomal recessive disordersAutosomal recessive disorders Risk calculationsRisk calculations

Specialist service design and provisionSpecialist service design and provision Choice of screening strategyChoice of screening strategy Problems and pitfallsProblems and pitfalls

Relevance of Relevance of ConsanguinityConsanguinity

Diverse populations served by each Diverse populations served by each lablab

Range of ethnic groups practice Range of ethnic groups practice consanguineous unionsconsanguineous unions

Consanguinity may alter testing Consanguinity may alter testing strategy and/or interpretation of strategy and/or interpretation of resultsresults

Testing for Autosomal Testing for Autosomal Recessive DisordersRecessive Disorders

Atypical, rare or private mutationAtypical, rare or private mutation Usual screening strategy may have lower Usual screening strategy may have lower

sensitivitysensitivity Negative result may not significantly Negative result may not significantly

reduce likelihood of diagnosisreduce likelihood of diagnosis Need for additional screening Need for additional screening

(availability/cost)(availability/cost) Affecteds expected to be homozygousAffecteds expected to be homozygous

Confirmation of homozygosityConfirmation of homozygosity Autozygosity analysis may be of useAutozygosity analysis may be of use

Cystic FibrosisCystic Fibrosis

Complex multi-system disorder that may Complex multi-system disorder that may affect the respiratory, pancreatic, gastro-affect the respiratory, pancreatic, gastro-intestinal and reproductive organ systems.intestinal and reproductive organ systems.

Incidence 1/2,500 (Caucasians)Incidence 1/2,500 (Caucasians) Less frequent in other populationsLess frequent in other populations

Carrier FrequencyCarrier Frequency 1/20 - 1/25 (Caucasians)1/20 - 1/25 (Caucasians)

Mutations in Mutations in CFTRCFTR gene gene >1600 different mutations reported>1600 different mutations reported

Cystic FibrosisCystic Fibrosis Initial screening for 29-32 mutationsInitial screening for 29-32 mutations

80-90% mutations (Caucasian)80-90% mutations (Caucasian) No mutation is detectedNo mutation is detected

Reduces likelihood (~2% affected CF patients Reduces likelihood (~2% affected CF patients would give this result)would give this result)

Single mutation detectedSingle mutation detected Increases likelihood (but doesn’t confirm)Increases likelihood (but doesn’t confirm)

However, if consanguineous:However, if consanguineous: % mutations detected <80% % mutations detected <80% Standard interpretation of negative result Standard interpretation of negative result

inaccurate inaccurate Potential homozygosity for rarer mutation in kitPotential homozygosity for rarer mutation in kit

Confirmation of atypical resultConfirmation of atypical result Testing of parental samplesTesting of parental samples

Interpretation of heterozygosityInterpretation of heterozygosity

Cystic FibrosisCystic Fibrosis Additional studies in consanguineous Additional studies in consanguineous

pedigreespedigrees Linked markersLinked markers

Autozygosity analysisAutozygosity analysis Support or exclude autozygous inheritance Support or exclude autozygous inheritance

Full sequencingFull sequencing Implications for neonatal screening Implications for neonatal screening

4 most common Caucasian mutations 4 most common Caucasian mutations

screened onlyscreened only 2nd raised IRT result for “high 2nd raised IRT result for “high

likelihood”/clinical referrallikelihood”/clinical referral

Spinal Muscular Atrophy Spinal Muscular Atrophy (SMA)(SMA)

Degeneration and loss of the proximal Degeneration and loss of the proximal anterior horn cells in the spinal cordanterior horn cells in the spinal cord Muscle wasting and atrophyMuscle wasting and atrophy

Incidence: 1/10,000Incidence: 1/10,000 Carrier frequency: 1/50Carrier frequency: 1/50 SMN1SMN1 gene gene >95% homozygous for deletion exon 7 >95% homozygous for deletion exon 7

(most exon 8 also deleted)(most exon 8 also deleted) Compound hets deletion/point mutationCompound hets deletion/point mutation

Spinal Muscular Atrophy Spinal Muscular Atrophy (SMA)(SMA)

First level test: screen for deletion of First level test: screen for deletion of SMN1SMN1 exon 7 and exon 8exon 7 and exon 8

If no deletion detected report as diagnosis If no deletion detected report as diagnosis “highly unlikely” or “extremely unlikely”“highly unlikely” or “extremely unlikely”

However, if consanguineousHowever, if consanguineous Possible homozygosity for point mutationPossible homozygosity for point mutation More cautious interpretationMore cautious interpretation ““reduces likelihood but cannot exclude a reduces likelihood but cannot exclude a

diagnosis”diagnosis” Linkage/autozygosity analysisLinkage/autozygosity analysis Screen for point mutations - no UK lab offering?Screen for point mutations - no UK lab offering?

Risk CalculationsRisk Calculations

Coefficient Inbreeding (F) - probability Coefficient Inbreeding (F) - probability that that childchild of consanguineous union will of consanguineous union will be homozygous for allele derived from be homozygous for allele derived from common ancestorcommon ancestor

Coefficient of Relationship (R) - proportion Coefficient of Relationship (R) - proportion of genes shared by related individuals of genes shared by related individuals

F = R x 1/2F = R x 1/2 If one parent transmits a particular allele If one parent transmits a particular allele

what is probability the other parent will what is probability the other parent will transmit same alleletransmit same allele


First Cousins:

R=1/8

First Cousins:

R=1/8

First Cousins:

R=1/8

First Cousins:

R=1/8


First Cousins Once Removed:

R=1/16


Second Cousins:

R=1/32


CF: No family historyNon-consanguineous = 1/22 x 1/22 x 1/4 = 1/1936


CF: No family historyConsanguineous = 1/22 x 1/8 x 1/4 = 1/704

R=1/8


Increased impact for rarer disordersIncreased impact for rarer disorders


Known carrier of CF mutation:Consanguineous = 1 x 1/8 x 1/4 = 1/32(Non-consanguineous = 1x 1/22 x 1/4 = 1/88)

R=1/8

Developing and Developing and Running a Running a

Diagnostic Service Diagnostic Service for Rare Recessive for Rare Recessive

DisordersDisorders

Assay DesignAssay Design

Gene structureGene structure Mutation spectrumMutation spectrum

Trinucleotide repeat expansionsTrinucleotide repeat expansions Large re-arrangementsLarge re-arrangements Point mutationsPoint mutations

Mutation distributionMutation distribution Recurrent mutations (founder effects)Recurrent mutations (founder effects) Mutation hot-spotsMutation hot-spots

Mutation ScanningMutation Scanning

Private mutationsPrivate mutations Whole gene screeningWhole gene screening

Scanning techniqueScanning technique Different behaviour of Different behaviour of

heteroduplexesheteroduplexes CSCECSCE dHPLCdHPLC HRMHRM

Heteroduplex AnalysisHeteroduplex Analysis

Denature & Re-anneal


Heteroduplex Analysis for Heteroduplex Analysis for Rare Recessive DisordersRare Recessive Disorders

ProblemProblem Reduced sensitivityReduced sensitivity

Sequence/mutation specific – ALMS1 Sequence/mutation specific – ALMS1 exampleexample



?

Heteroduplex Analysis - Heteroduplex Analysis - Reduced Sensitivity for Reduced Sensitivity for Homozygous ChangesHomozygous Changes HRM analysisHRM analysis

Blind trial of 14 previously tested patientsBlind trial of 14 previously tested patients 10 different amplicons10 different amplicons

Each patient had mutation or variant in at least Each patient had mutation or variant in at least one amplicon but not variant for each ampliconone amplicon but not variant for each amplicon

3 false negatives3 false negatives All 3 were homozygous changesAll 3 were homozygous changes Other homozygous changes were detectedOther homozygous changes were detected Dependent on nature of variant and sequence Dependent on nature of variant and sequence

contextcontext




SolutionsSolutions Screen parentsScreen parents Spiking – ALMS1 exampleSpiking – ALMS1 example

Spiking of PCRs with Spiking of PCRs with Wildtype DNAWildtype DNA

To enable detection of homozygote To enable detection of homozygote variants by facilitating heteroduplex variants by facilitating heteroduplex formationformation

Pre-PCR spikingPre-PCR spiking Post-PCR spikingPost-PCR spiking

Visualise amplification of all samplesVisualise amplification of all samples Analyse samples before and after Analyse samples before and after

spiking spiking (prevent missing heterozygous (prevent missing heterozygous changes masked by WT alleles)changes masked by WT alleles)

Spiking of PCRs with Spiking of PCRs with Wildtype DNAWildtype DNA

Test samples (incl. blank & controls)1.5xreaction volume

Wildtype controls1.5xreaction volume

1/3 vol. test and wildtype(all 3 columns equal vol.)Denature & re-anneal, then analyse (1 v 2 and 3 v 2)




Solutions Solutions Screen parentsScreen parents Spiking – ALMS1 exampleSpiking – ALMS1 example

Large number of WT controls per run Large number of WT controls per run (cost and DNA availability)(cost and DNA availability) Lack of batching (need group of WT samples Lack of batching (need group of WT samples

to give normal pattern to compare against)to give normal pattern to compare against)

Interpretation of ResultsInterpretation of Results Pathogenicity of private mutationPathogenicity of private mutation

Limited mutations publishedLimited mutations published Less functional dataLess functional data Fewer orthologuesFewer orthologues

HeterozyoteHeterozyote Possibility of two different mutations within Possibility of two different mutations within

familyfamily Genetic heterogeneityGenetic heterogeneity

Possibility of mutation in two different genes Possibility of mutation in two different genes giving similar phenotype in familygiving similar phenotype in family

ConclusionsConclusions

Consanguinity is likely to be encountered Consanguinity is likely to be encountered by all diagnostic laboratories.by all diagnostic laboratories.

Awareness of consanguinity important to Awareness of consanguinity important to enable provision of suitable tests and enable provision of suitable tests and accurate interpretation of results.accurate interpretation of results.

Consanguinity has effect on risk Consanguinity has effect on risk calculations.calculations.

Services for rare recessive disorders must Services for rare recessive disorders must be designed to detect homozygous be designed to detect homozygous variants with high sensitivity.variants with high sensitivity.

Documents

Implications of Consanguinity for Routine Diagnostic Testing and Development of Specialist Services Teresa Lamb Clinical Scientist Leeds DNA Laboratory